Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same

ABSTRACT

A mixture of conjugates in which each conjugate in the mixture comprises an insulin drug coupled to an oligomer that includes a polyalkylene glycol moiety is disclosed. The mixture may exhibit higher in vivo activity than a polydispersed mixture of similar conjugates. The mixture may also be more effective at surviving an in vitro model of intestinal digestion than polydispersed mixtures of similar conjugates. The mixture may also result in less inter-subject variability than polydispersed mixtures of similar conjugates.

RELATED APPLICATION

[0001] This application is a continuation of, and claims priority to,U.S. patent application Ser. No. 09/873,899, filed Jun. 4, 2001, nowallowed, assigned to the assignee of the present invention, thedisclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to drug-oligomer conjugates, and,more particularly, to insulin drug-oligomer conjugates.

BACKGROUND OF THE INVENTION

[0003] Diabetes, a disorder of carbohydrate metabolism, has been knownsince antiquity. Diabetes results from insufficient production of orreduced sensitivity to insulin. The insulin molecule consists of twochains of amino acids linked by disulfide bonds (mw 6,000). The β-cellsof the pancreatic islets secrete a single chain precursor of insulin,known as proinsulin. Proteolysis of proinsulin results in removal offour basic amino acids (numbers 31, 32, 64 and 65 in the proinsulinchain: Arg, Arg, Lys, Arg respectively) and the connecting (“C”)polypeptide. In the resulting two-chain insulin molecule, the A chainhas glycine at the amino terminus, and the B chain has phenylalanine atthe amino terminus.

[0004] Insulin may exist as a monomer, dimer or a hexamer formed fromthree of the dimers. The hexamer is coordinated with two Zn²⁺ atoms.Biological activity resides in the monomer. Although until recentlybovine and porcine insulin were used almost exclusively to treatdiabetes in humans, numerous variations in insulin between species areknown. Porcine insulin is most similar to human insulin, from which itdiffers only in having an alanine rather than threonine residue at theB-chain C-terminus. Despite these differences most mammalian insulin hascomparable specific activity. Until recently animal extracts providedall insulin used for treatment of the disease. The advent of recombinanttechnology commercially available from Eli Lilly and Company,Indianapolis, Ind.).

[0005] Insulin is necessary for normal utilization of glucose by mostcells in the body. In persons with diabetes, the normal ability to useglucose is inhibited, thereby increasing blood sugar levels(hyperglycemia). As glucose accumulates in the blood, excess levels ofsugar are excreted in the urine (glycosuria). Other symptoms of diabetesinclude increased urinary volume and frequency, thirst, itching, hunger,weight loss, and weakness.

[0006] There are two varieties of diabetes. Type I is insulin-dependentdiabetes mellitus, or IDDM. IDDM was formerly referred to as juvenileonset diabetes. In IDDM, insulin is not secreted by the pancreas andmust be provided from an external source. Type II adult-onset diabetescan ordinarily be controlled by diet although in some advanced casesinsulin is required.

[0007] Before the isolation of insulin in the 1920s, most patients diedwithin a short time after onset. Untreated diabetes leads to ketosis,the accumulation of ketones, products of fat breakdown, in the blood;this is followed by acidosis (accumulation of acid in the blood) withnausea and vomiting. As the toxic products of disordered carbohydrateand fat metabolism continue to build up, the patient goes into diabeticcoma.

[0008] Treatment of diabetes typically requires regular injections ofinsulin. The use of insulin as a treatment for diabetes dates to 1922,when Banting et al. (“Pancreatic Extracts in the Treatment of DiabetesMellitus,” Can. Med. Assoc. J., 12: 141-146 (1922)) showed that theactive extract from the pancreas had therapeutic effects in diabeticdogs. Treatment of a diabetic patient in that same year with pancreaticextracts resulted in a dramatic, life-saving clinical improvement. Dueto the inconvenience of insulin injections, massive efforts to improveinsulin administration and bioassimilation have been undertaken.

[0009] Attempts have been made to deliver insulin by oraladministration. The problems associated with oral administration ofinsulin to achieve euglycemia in diabetic patients are well documentedin pharmaceutical and medical literature. Digestive enzymes in the GItract rapidly degrade insulin, resulting in biologically inactivebreakdown products. In the stomach, for example, orally administeredinsulin undergoes enzymatic proteolysis and acidic degradation. Survivalin the intestine is hindered by excessive proteolysis. In the lumen,insulin is barraged by a variety of enzymes including gastric andpancreatic enzymes, exo- and endopeptidases, and brush borderpeptidases. Even if insulin survives this enzymatic attack, thebiological barriers that must be traversed before insulin can reach itsreceptors in vivo may limit oral administration of insulin. For example,insulin may possess low membrane permeability, limiting its ability topass from the lumen into the bloodstream.

[0010] Pharmaceutically active polypeptides such as insulin have beenconjugated with polydispersed mixtures of polyethylene glycol orpolydispersed mixtures of polyethylene glycol containing polymers toprovide polydispersed mixtures of drug-oligomer conjugates. For example,U.S. Pat. No. 4,179,337 to Davis et al. proposes conjugatingpolypeptides such as insulin with various polyethylene glycols such asMPEG-1900 and MPEG-5000 supplied by Union Carbide.

[0011] U.S. Pat. No. 5,567,422 to Greenwald proposes the conjugation ofbiologically active nucleophiles with polyethylene glycols such asm-PEG-OH (Union Carbide), which has a number average molecular weight of5,000 Daltons.

[0012] U.S. Pat. No. 5,359,030 to Ekwuribe proposes conjugatingpolypeptides such as insulin with polyethylene glycol modifiedglycolipid polymers and polyethylene glycol modified fatty acidpolymers. The number average molecular weight of polymer resulting fromeach combination is preferred to be in the range of from about 500 toabout 10,000 Daltons.

[0013] Polyethylene glycol is typically produced by base-catalyzedring-opening polymerization of ethylene oxide. The reaction is initiatedby adding ethylene oxide to ethylene glycol, with potassium hydroxide ascatalyst. This process results in a polydispersed mixture ofpolyethylene glycol polymers having a number average molecular weightwithin a given range of molecular weights. For example, PEG productsoffered by Sigma-Aldrich of Milwaukee, Wis. in are provided inpolydispersed mixtures such as PEG 400 (M_(n) 380-420); PEG 1,000 (M_(n)950-1,050); PEG 1,500 (M_(n) 1,400-1,600); and PEG 2,000 (M_(n)1,900-2,200).

[0014] It is desirable to provide non-polydispersed mixtures ofinsulin-oligomer conjugates where the oligomer comprises polyethyleneglycol.

SUMMARY OF THE INVENTION

[0015] It has unexpectedly been discovered that a non-polydispersedmixture of insulin-oligomer conjugates comprising polyethylene glycolaccording to embodiments of the present invention exhibits higher invivo activity than a polydispersed mixture of similar conjugates havingthe same number average molecular weight. This heightened activity mayresult in lower dosage requirements. Moreover, a non-polydispersedmixture of insulin-oligomer conjugates comprising polyethylene glycolaccording to embodiments of the present invention is typically moreeffective at surviving an in vitro model of intestinal digestion thanpolydispersed mixtures of similar conjugates. Furthermore,non-polydispersed mixtures of insulin-oligomer conjugates comprisingpolyethylene glycol according to embodiments of the present inventionalso typically result in less inter-subject variability thanpolydispersed mixtures of similar conjugates.

[0016] According to embodiments of the present invention, asubstantially monodispersed mixture of conjugates each comprising aninsulin drug coupled to an oligomer that comprises a polyethylene glycolmoiety is provided. The polyethylene glycol moiety preferably has atleast two, three, or four polyethylene glycol subunits and, mostpreferably, has at least seven polyethylene glycol subunits. Theoligomer preferably further comprises a lipophilic moiety. The insulindrug is preferably human insulin. The oligomer is preferably covalentlycoupled to Lys^(B29) of the human insulin. The conjugate is preferablyamphiphilically balanced such that the conjugate is aqueously solubleand able to penetrate biological membranes. The mixture is preferably amonodispersed mixture and is most preferably a purely monodispersedmixture. In some embodiments, the oligomer comprises a firstpolyethylene glycol moiety covalently coupled to the insulin by anon-hydrolyzable bond and a second polyethylene glycol moiety covalentlycoupled to the first polyethylene glycol moiety by a hydrolyzable bond.

[0017] According to other embodiments of the present invention, asubstantially monodispersed mixture of conjugates is provided where eachconjugate includes human insulin covalently coupled at LyS^(B29) of thehuman insulin to a carboxylic acid moiety of an oligomer that compriseshexanoic acid covalently coupled at the end distal to the carboxylicacid moiety to a methyl terminated polyethylene glycol moiety having atleast 7 polyethylene glycol subunits.

[0018] Substantially monodispersed mixtures of conjugates according tothese embodiments of the present invention preferably have improvedproperties when compared with those of polydispersed mixtures. In oneembodiment, a substantially monodispersed mixture of conjugates isprovided where each conjugate comprises an insulin drug coupled to anoligomer including a polyethylene glycol moiety, and the mixture has anin vivo activity that is greater than the in vivo activity of apolydispersed mixture of insulin drug-oligomer conjugates having thesame number average molecular weight as the substantially monodispersedmixture.

[0019] In another embodiment, a substantially monodispersed mixture ofconjugates is provided where each conjugate comprises an insulin drugcoupled to an oligomer including a polyethylene glycol moiety, and themixture has an in vitro activity that is greater than the in vitroactivity of a polydispersed mixture of insulin drug-oligomer conjugateshaving the same number average molecular weight as the substantiallymonodispersed mixture.

[0020] In still another embodiment, a substantially monodispersedmixture of conjugates is provided where each conjugate comprises aninsulin drug coupled to an oligomer including a polyethylene glycolmoiety, and the mixture has an increased resistance to degradation bychymotrypsin when compared to the resistance to degradation bychymotrypsin of a polydispersed mixture of insulin drug-oligomerconjugates having the same number average molecular weight as thesubstantially monodispersed mixture.

[0021] In yet another embodiment, a substantially monodispersed mixtureof conjugates is provided where each conjugate comprises an insulin drugcoupled to an oligomer including a polyethylene glycol moiety, and themixture has an inter-subject variability that is less than theinter-subject variability of a polydispersed mixture of insulindrug-oligomer conjugates having the same number average molecular weightas the substantially monodispersed mixture.

[0022] Substantially monodispersed mixtures of conjugates according toembodiments of the present invention preferably have two or more of theabove-described properties. More preferably, substantially monodispersedmixtures of conjugates according to embodiments of the present inventionhave three or more of the above-described properties. Most preferably,substantially monodispersed mixtures of conjugates according toembodiments of the present invention have all four of theabove-described properties.

[0023] According to still other embodiments of the present invention, amixture of conjugates is provided where each conjugate includes aninsulin drug coupled to an oligomer that comprises a polyethylene glycolmoiety, and the mixture has a molecular weight distribution with astandard deviation of less than about 22 Daltons.

[0024] According to yet other embodiments of the present invention, amixture of conjugates is provided where each conjugate includes aninsulin drug coupled to an oligomer that comprises a polyethylene glycolmoiety, and the mixture has a dispersity coefficient (DC) greater than10,000 where${D\quad C} = \frac{\left( {\sum\limits_{i = 1}^{n}{N_{i}M_{i}}} \right)^{2}}{{\sum\limits_{i = 1}^{n}{N_{i}M_{i}^{2}{\sum\limits_{i = 1}^{n}N_{i}}}} - \left( {\sum\limits_{i = 1}^{n}{N_{i}M_{i}}} \right)^{2}}$

[0025] wherein:

[0026] n is the number of different molecules in the sample;

[0027] N_(i) is the number of i^(th) molecules in the sample; and

[0028] M_(i) is the mass of the i^(th) molecule.

[0029] According to other embodiments of the present invention, amixture of conjugates is provided in which each conjugate includes aninsulin drug coupled to an oligomer and has the same number ofpolyethylene glycol subunits.

[0030] According to still other embodiments of the present invention, amixture of conjugates is provided in which each conjugate is the sameand has the formula:

Insulin DrugB-L_(j)-G_(k)-R-G′_(m)-R′-G″_(n)-T]_(p)   (A)

[0031] wherein:

[0032] B is a bonding moiety;

[0033] L is a linker moiety;

[0034] G, G′ and G″ are individually selected spacer moieties;

[0035] R is a lipophilic moiety and R′ is a polyalkylene glycol moiety,or R′ is the lipophilic moiety and R is the polyalkylene glycol moiety;

[0036] T is a terminating moiety;

[0037] j, k, m and n are individually 0 or 1; and

[0038] p is an integer between 1 and the number of nucleophile residueson the insulin drug.

[0039] Pharmaceutical compositions comprising conjugate mixtures of thepresent invention as well as methods of treating an insulin deficiencyin a subject in need of such treatment by administering an effectiveamount of such pharmaceutical compositions are also provided.Additionally, methods of synthesizing such conjugate mixtures areprovided.

[0040] Insulin-oligomer conjugate mixtures according to embodiments ofthe present invention may provide increased in vivo activity and/orlowered inter-subject variability and/or decreased degradation bychymotrypsin when compared to conventional polydispersedinsulin-oligomer conjugate mixtures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 illustrates a generic scheme for synthesizing a mixture ofactivated polymers comprising a polyethylene glycol moiety and a fattyacid moiety according to embodiments of the present invention;

[0042]FIG. 2 illustrates a scheme for synthesizing a mixture of mPEGaccording to embodiments of the present invention;

[0043]FIG. 3 illustrates a scheme for synthesizing a mixture ofactivated mPEG7-hexyl oligomers according to embodiments of the presentinvention;

[0044]FIG. 4 illustrates a scheme for synthesizing a mixture ofactivated mPEG7-octyl oligomers according to embodiments of the presentinvention;

[0045]FIG. 5 illustrates a scheme for synthesizing a mixture ofactivated mPEG-decyl oligomers according to embodiments of the presentinvention;

[0046]FIG. 6 illustrates a scheme for synthesizing a mixture ofactivated stearate-PEG6 oligomers according to embodiments of thepresent invention;

[0047]FIG. 7 illustrates a scheme for synthesizing a mixture ofactivated stearate-PEG8 oligomers according to embodiments of thepresent invention;

[0048]FIG. 8 illustrates a scheme for synthesizing a mixture ofactivated PEG3 oligomers according to embodiments of the presentinvention;

[0049]FIG. 9 illustrates a scheme for synthesizing a mixture ofactivated palmitate-PEG3 oligomers according to embodiments of thepresent invention;

[0050]FIG. 10 illustrates a scheme for synthesizing a mixture ofactivated PEG6 oligomers according to embodiments of the presentinvention;

[0051]FIG. 11 illustrates a scheme for synthesizing various propyleneglycol monomers according to embodiments of the present invention;

[0052]FIG. 12 illustrates a scheme for synthesizing various propyleneglycol polymers according to embodiments of the present invention;

[0053]FIG. 13 illustrates a scheme for synthesizing various propyleneglycol polymers according to embodiments of the present invention;

[0054]FIG. 14 illustrates a comparison of results obtained with aCytosensor® Microphysiometer, which provides an indication of theactivity of a compound, for mixtures of insulin-oligomer conjugatesaccording to embodiments of the present invention compared withpolydispersed conjugate mixtures and insulin, which are provided forcomparison purposes only and do not form part of the invention;

[0055]FIG. 15 illustrates a comparison of chymotrypsin degradation ofinsulin-oligomer conjugates according to embodiments of the presentinvention with a conventional polydispersed mixture of insulin-oligomerconjugates, which is provided for comparison purposes only and does riotform part of the invention;

[0056]FIG. 16 illustrates the effect of a mixture ofmPEG7-hexyl-insulin, monoconjugate, according to embodiments of thepresent invention on plasma glucose in fasted beagles;

[0057]FIG. 17 illustrates, for comparison purposes, the effect of apolydispersed mixture of mPEG7_(avg)-hexyl-insulin, monoconjugate, whichis not part of the present invention, on plasma glucose in fastedbeagles;

[0058]FIG. 18 illustrates the inter-subject variability of a mixture ofmPEG4-hexyl-insulin monoconjugates according to embodiments of thepresent invention administered to fasted beagles;

[0059]FIG. 19 illustrates the inter-subject variability of a mixture ofmPEG7-hexyl-insulin monoconjugates according to embodiments of thepresent invention administered to fasted beagles;

[0060]FIG. 20 illustrates the inter-subject variability for a mixture ofmPEG10-hexyl-insulin monoconjugates according to embodiments of thepresent invention administered to fasted beagles; and

[0061]FIG. 21 illustrates, for comparison purposes, the inter-subjectvariability of a polydispersed mixture of mPEG7_(avg)-hexyl-insulinmonoconjugates, which is not part of the present invention, administeredto fasted beagles.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0062] The invention will now be described with respect to preferredembodiments described herein. It should be appreciated however thatthese embodiments are for the purpose of illustrating the invention, andare not to be construed as limiting the scope of the invention asdefined by the claims.

[0063] As used herein, the term “non-polydispersed” is used to describea mixture of compounds having a dispersity that is in contrast to thepolydispersed mixtures described in U.S. Pat. No. 4,179,337 to Davis etal.; U.S. Pat. No. 5,567,422 to Greenwald; U.S. Pat. No. 5,405,877 toGreenwald et al.; and U.S. Pat. No. 5,359,030 to Ekwuribe.

[0064] As used herein, the term “substantially monodispersed” is used todescribe a mixture of compounds wherein at least about 95 percent of thecompounds in the mixture have the same molecular weight.

[0065] As used herein, the term “monodispersed” is used to describe amixture of compounds wherein about 100 percent of the compounds in themixture have the same molecular weight.

[0066] As used herein, the term “substantially purely monodispersed” isused to describe a mixture of compounds wherein at least about 95percent of the compounds in the mixture have the same molecular weightand have the same molecular structure. Thus, a substantially purelymonodispersed mixture is a substantially monodispersed mixture, but asubstantially monodispersed mixture is not necessarily a substantiallypurely monodispersed mixture.

[0067] As used herein, the term “purely monodispersed” is used todescribe a mixture of compounds wherein about 100 percent of thecompounds in the mixture have the same molecular weight and have thesame molecular structure. Thus, a purely monodispersed mixture is amonodispersed mixture, but a monodispersed mixture is not necessarily apurely monodispersed mixture.

[0068] As used herein, the term “weight average molecular weight” isdefined as the sum of the products of the weight fraction for a givenmolecule in the mixture times the mass of the molecule for each moleculein the mixture. The “weight average molecular weight” is represented bythe symbol M_(w).

[0069] As used herein, the term “number average molecular weight” isdefined as the total weight of a mixture divided by the number ofmolecules in the mixture and is represented by the symbol M_(n).

[0070] As used herein, the term “dispersity coefficient” (DC) is definedby the formula:${D\quad C} = \frac{\left( {\sum\limits_{i = 1}^{n}{N_{i}M_{i}}} \right)^{2}}{{\sum\limits_{i = 1}^{n}{N_{i}M_{i}^{2}{\sum\limits_{i = 1}^{n}N_{i}}}} - \left( {\sum\limits_{i = 1}^{n}{N_{i}M_{i}}} \right)^{2}}$

[0071] wherein:

[0072] n is the number of different molecules in the sample;

[0073] N_(i) is the number of i^(th) molecules in the sample; and

[0074] M_(i) is the mass of the i^(th) molecule.

[0075] As used herein, the term “intra-subject variability” means thevariability in activity occurring within the same subject when thesubject is administered the same dose of a drug or pharmaceuticalcomposition at different times.

[0076] As used herein, the term “inter-subject variability” means thevariability in activity between two or more subjects when each subjectis administered the same dose of a given drug or pharmaceuticalformulation.

[0077] As used herein, the term “insulin drug” means a drug possessingall or some of the biological activity of insulin.

[0078] As used herein, the term “insulin” means human insulin, bovineinsulin,.porcine insulin or whale insulin, provided by natural,synthetic, or genetically engineered sources.

[0079] As used herein, the term “insulin analog” means insulin whereinone or more of the amino acids have been replaced while retaining someor all of the activity of the insulin. The analog is described by notingthe replacement amino acids with the position of the replacement as asuperscript followed by a description of the insulin. For example,“Pro^(B29) insulin, human” means that the lysine typically found at theB29 position of a human insulin molecule has been replaced with proline.

[0080] Insulin analogs may be obtained by various means, as will beunderstood by those skilled in the art. For example, certain amino acidsmay be substituted for other amino acids in the insulin structurewithout appreciable loss of interactive binding capacity with structuressuch as, for example, antigen-binding regions of antibodies or bindingsites on substrate molecules. As the interactive capacity-and nature ofinsulin defines its biological functional activity, certain amino acidsequence substitutions can be made in the amino acid sequence andnevertheless remain a polypeptide with like properties.

[0081] In making such substitutions, the hydropathic index of aminoacids may be considered. The importance of the hydropathic amino acidindex in conferring interactive biologic function on a polypeptide isgenerally understood in the art. It is accepted that the relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant polypeptide, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Eachamino acid has been assigned a hydropathic index on the basis of itshydrophobicity and charge characteristics as follows: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine(+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine(−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline(−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate(−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). As willbe understood by those skilled in the art, certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity, i.e., still obtain a biological functionally equivalentpolypeptide. In making such changes, the substitution of amino acidswhose hydropathic indices are within ±2 of each other is preferred,those which are within ±1 of each other are particularly preferred, andthose within ±0.5 of each other are even more particularly preferred.

[0082] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101 provides that the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with a biological property of theprotein. As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (±3.0); aspartate (+3.0±1); glutamate (+3.0±1);seine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−9.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4). As is understood by those skilled in the art, an amino acid canbe substituted for another having a similar hydrophilicity value andstill obtain a biologically equivalent, and in particular, animmunologically equivalent polypeptide. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ofeach other is preferred, those which are within ±1 of each other areparticularly preferred, and those within ±0.5 of each other are evenmore particularly preferred.

[0083] As outlined above, amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions (i.e., amino acids that maybe interchanged without significantly altering the biological activityof the polypeptide) that take various of the foregoing characteristicsinto consideration are well known to those of skill in the art andinclude, for example: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

[0084] As used herein, the term “insulin fragment” means a segment ofthe amino acid sequence found in the insulin that retains some or all ofthe activity of the insulin. Insulin fragments are denoted by statingthe position(s) in an amino acid sequence followed by a description ofthe amino acid. For example, a “B25-B30 human insulin” fragment would bethe six amino acid sequence corresponding to the B25, B26, B27, B28, B29and B30 positions in the human insulin amino acid sequence.

[0085] As used herein, the term “insulin fragment analog” means asegment of the amino acid sequence found in the insulin molecule whereinone or more of the amino acids in the segment have been replace whileretaining some or all of the activity of the insulin.

[0086] As used herein, the term “PEG” refers to straight or branchedpolyethylene glycol polymers, and includes the monomethylether ofpolyethylene glycol (mPEG). The terms “PEG subunit” and polyethyleneglycol subunit refer to a single polyethylene glycol unit, i.e.,—(CH₂CH₂O)—.

[0087] As used herein, the term “lipophilic” means the ability todissolve in lipids and/or the ability to penetrate, interact with and/ortraverse biological membranes, and the term, “lipophilic moiety” or“lipophile” means a moiety which is lipophilic and/or which, whenattached to another chemical entity, increases the lipophilicity of suchchemical entity. Examples of lipophilic moieties include, but are notlimited to, alkyls, fatty acids, esters of fatty acids, cholesteryl,adamantyl and the like.

[0088] As used herein, the term “lower alkyl” refers to substituted orunsubstituted alkyl moieties having from one to five carbon atoms.

[0089] As used herein, the term “higher alkyl” refers to substituted orunsubstituted alkyl moieties having six or more carbon atoms.

[0090] In embodiments of the present invention, a substantiallymonodispersed mixture of insulin-oligomer conjugates is provided.Preferably, at least about 96, 97, 98 or 99 percent of the conjugates inthe mixture have the same molecular weight. More preferably, the mixtureis a monodispersed mixture. Even more preferably, the mixture is asubstantially purely monodispersed mixture. Still more preferably, atleast about 96, 97, 98 or 99 percent of the conjugates in the mixturehave the same molecular weight and have the same molecular structure.Most preferably, the mixture is a purely monodispersed mixture.

[0091] The insulin drug is preferably insulin. More preferably, theinsulin drug is human insulin. However, it is to be understood that theinsulin drug may be selected from various insulin drugs known to thoseskilled in the art including, for example, proinsulin, insulin analogs,insulin fragments, and insulin fragment analogs. Insulin analogsinclude, but are not limited to, Asp^(B28) human insulin, Lys^(B28)human insulin, Leu^(B28) human insulin, Val^(B28) human insulin,Ala^(B28) human insulin, ASP^(B28)Pro^(B29) human insulin,Lys^(B28)Pro^(B29) human insulin, Leu^(B28)Pro^(B29) human insulin,Val^(B28)Pro^(B29) human insulin, Ala^(B28)Pro^(B29) human insulin, aswell as analogs provided using the substitution guidelines describedabove. Insulin fragments include, but are not limited to, B22-B30 humaninsulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 humaninsulin, B27-B30 human insulin, B29-B30 human insulin, the A chain ofhuman insulin, and the B chain of human insulin. Insulin fragmentanalogs may be provided by substituting one or more amino acids asdescribed above in an insulin fragment.

[0092] The oligomer may be various oligomers comprising a polyethyleneglycol moiety as will be understood by those skilled in the art.Preferably, the polyethylene glycol moiety of the oligomer has at least2, 3 or 4 polyethylene glycol subunits. More preferably, thepolyethylene glycol moiety has at least 5 or 6 polyethylene glycolsubunits and, most preferably, the polyethylene glycol moiety has atleast 7 polyethylene glycol subunits.

[0093] The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,additional hydrophilic moieties, lipophilic moieties, spacer moieties,linker moieties, and terminating moieties. The various moieties in theoligomer are covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

[0094] The oligomer may further comprise one or more additionalhydrophilic moieties (i.e., moieties in addition to the polyethyleneglycol moiety) including, but not limited to, sugars, polyalkyleneoxides, and polyamine/PEG copolymers. As polyethylene glycol is apolyalkylene oxide, the additional hydrophilic moiety may be apolyethylene glycol moiety. Adjacent polyethylene glycol moieties willbe considered to be the same moiety if they are coupled by an etherbond. For example, the moiety

—O—C₂H₄—O—C₂H₄ —O—C₂H₄—C₂H₄—O—C₂H₄ —C₂H₄—

[0095] is a single polyethylene glycol moiety having six polyethyleneglycol subunits. If this moiety were the only hydrophilic moiety in theoligomer, the oligomer would not contain an additional hydrophilicmoiety. Adjacent polyethylene glycol moieties will be considered to bedifferent moieties if they are coupled by a bond other than an etherbond. For example, the moiety

[0096] is a polyethylene glycol moiety having four polyethylene glycolsubunits and an additional hydrophilic moiety having two polyethyleneglycol subunits. Preferably, oligomers according to embodiments of thepresent invention comprise a polyethylene glycol moiety and noadditional hydrophilic moieties.

[0097] The oligomer may further comprise one or more lipophilic moietiesas will be understood by those skilled in the art. The lipophilic moietyis preferably a saturated or unsaturated, linear or branched alkylmoiety or a saturated or unsaturated, linear or branched fatty acidmoiety. When the lipophilic moiety is an alkyl moiety, it is preferablya linear, saturated or unsaturated alkyl moiety having 1 to 28 carbonatoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. Whenthe lipophilic moiety is a fatty acid moiety, it is preferably a naturalfatty acid moiety that is linear, saturated or unsaturated, having 2 to18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5or 6 carbon atoms.

[0098] The oligomer may further comprise, one or more spacer moieties aswill be understood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from theinsulin drug, to separate a first hydrophilic or lipophilic moiety froma second hydrophilic or lipophilic moiety, or to separate a hydrophilicmoiety or lipophilic moiety from a linker moiety. Spacer moieties arepreferably selected from the group consisting of sugar, cholesterol andglycerine moieties.

[0099] The oligomer may further comprise one or more linker moietiesthat are used to couple the oligomer with the insulin drug as will beunderstood by those skilled in the art. Linker moieties are preferablyselected from the group consisting of alkyl and fatty acid moieties.

[0100] The oligomer may further comprise one or more terminatingmoieties at the one or more ends of the oligomer which are not coupledto the insulin drug. The terminating moiety is preferably an alkyl oralkoxy moiety, and is more preferably a lower alkyl or lower alkoxymoiety. Most preferably, the terminating moiety is methyl or methoxy.While the terminating moiety is preferably an alkyl or alkoxy moiety, itis to be understood that the terminating moiety may be various moietiesas will be understood by those skilled in the art including, but notlimited to, sugars, cholesterol, alcohols, and fatty acids.

[0101] The oligomer is preferably covalently coupled to the insulindrug. In some embodiments, the insulin drug is coupled to the oligomerutilizing a hydrolyzable bond (e.g., an ester or carbonate bond). Ahydrolyzable coupling may provide an insulin drug-oligomer conjugatethat acts as a prodrug. In certain instances, for example where theinsulin drug-oligomer conjugate is inactive (i.e., the conjugate lacksthe ability to affect the body through the insulin drug's primarymechanism of action), a hydrolyzable coupling may provide for atime-release or controlled-release effect, administering the insulindrug over a given time period as one or more oligomers are cleaved fromtheir respective insulin drug-oligomer conjugates to provide the activedrug. In other embodiments, the insulin drug is coupled to the oligomerutilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or etherbond). Use of a non-hydrolyzable bond may be preferable when it isdesirable to allow the insulin drug-oligomer conjugate to circulate inthe bloodstream for an extended period of time, preferably at least 2hours. When the oligomer is covalently coupled to the insulin drug, theoligomer further comprises one or more bonding moieties that are used tocovalently couple the oligomer with the insulin drug as will beunderstood by those skilled in the art. Bonding moieties are preferablyselected from the group consisting of covalent bond(s), ester moieties,carbonate moieties, carbamate moieties, amide moieties and secondaryamine moieties. More than one moiety on the oligomer may be covalentlycoupled to the insulin drug.

[0102] While the oligomer is preferably covalently coupled to theinsulin drug, it is to be understood that the oligomer may benon-covalently coupled to the insulin drug to form a non-covalentlyconjugated insulin drug-oligomer complex. As will be understood by thoseskilled in the art, non-covalent couplings include, but are not limitedto, hydrogen bonding, ionic bonding, Van der Waals bonding, andmicellular or liposomal encapsulation. According to embodiments of thepresent invention, oligomers may be suitably constructed, modifiedand/or appropriately functionalized to impart the ability fornon-covalent conjugation in a selected manner (e.g., to impart hydrogenbonding capability), as will be understood by those skilled in the art.According to other embodiments of present invention, oligomers may bederivatized with various compounds including, but not limited to, aminoacids, oligopeptides, peptides, bile acids, bile acid derivatives, fattyacids, fatty acid derivatives, salicylic acids, salicylic acidderivatives, aminosalicylic acids, and aminosalicylic acid derivatives.The resulting oligomers can non-covalently couple (complex) with drugmolecules, pharmaceutical products, and/or pharmaceutical excipients.The resulting complexes preferably have balanced lipophilic andhydrophilic properties. According to still other embodiments of thepresent invention, oligomers may be derivatized with amine and/or alkylamines. Under suitable acidic conditions, the resulting oligomers canform non-covalently conjugated complexes with drug molecules,pharmaceutical products and/or pharmaceutical excipients. The productsresulting from such complexation preferably have balanced lipophilic andhydrophilic properties.

[0103] More than one oligomer (i.e., a plurality of oligomers) may becoupled to the insulin drug. The oligomers in the plurality arepreferably the same. However, it is to be understood that the oligomersin the plurality may be different from one another, or, alternatively,some of the oligomers in the plurality may be the same and some may bedifferent. When a plurality of oligomers are coupled to the insulindrug, it may be preferable to couple one or more of the oligomers to theinsulin drug with hydrolyzable bonds and couple one or more of theoligomers to the insulin drug with non-hydrolyzable bonds.Alternatively, all of the bonds coupling the plurality of oligomers tothe insulin drug may be hydrolyzable, but have varying degrees ofhydrolyzability such that, for example, one or more of the oligomers israpidly removed from the insulin drug by hydrolysis in the body and oneor more of the oligomers is slowly removed from the insulin drug byhydrolysis in the body.

[0104] The oligomer may be coupled to the insulin drug at variousnucleophile residues of the insulin drug including, but not limited to,nucleophilic hydroxyl functions and/or amino functions. When the insulindrug is a polypeptide, a nucleophilic hydroxyl function may be found,for example, at serine and/or tyrosine residues, and a nucleophilicamino function may be found, for example, at histidine and/or lysineresidues, and/or at the one or more N-termini of the polypeptide. Whenan oligomer is coupled to the one or more N-termini of the insulinpolypeptide, the coupling preferably forms a secondary amine. When theinsulin drug is human insulin, for example, the oligomer may be coupledto an amino functionality of the insulin, including the aminofunctionality of Gly^(A1), the amino functionality of Phe^(B1), and theamino functionality of Lys^(B29). When one oligomer is coupled to thehuman insulin, the oligomer is preferably coupled to the aminofunctionality of Lys^(B29) When two oligomers are coupled to the humaninsulin, the oligomers are preferably coupled to the amino functionalityof Phe^(B1) and the amino functionality of Lys^(B29). While more thanone oligomer may be coupled to the human insulin, a higher activity(improved glucose lowering ability) is observed for the mono-conjugatedhuman insulin.

[0105] Substantially monodispersed mixtures of insulin drug-oligomerconjugates of the present invention may be synthesized by variousmethods. For example, a substantially monodispersed mixture of oligomersconsisting of carboxylic acid and polyethylene glycol is synthesized bycontacting a substantially monodispersed mixture of carboxylic acid witha substantially monodispersed mixture of polyethylene glycol underconditions sufficient to provide a substantially monodispersed mixtureof oligomers. The oligomers of the substantially monodispersed mixtureare then activated so that they are capable of reacting with an insulindrug to provide an insulin drug-oligomer conjugate. One embodiment of asynthesis route for providing a substantially monodispersed mixture ofactivated oligomers is illustrated in FIG. 3 and described in Examples11-18 hereinbelow. Another embodiment of a synthesis route for providinga substantially monodispersed mixture of activated oligomers isillustrated in FIG. 4 and described in Examples 19-24 hereinbelow. Stillanother embodiment of a synthesis route for providing a substantiallymonodispersed mixture of activated oligomers is illustrated in FIG. 5and described in Examples 25-29 hereinbelow. Yet another embodiment of asynthesis route for providing a substantially monodispersed mixture ofactivated oligomers is illustrated in FIG. 6 and described in Examples30-31 hereinbelow. Another embodiment of a synthesis route for providinga substantially monodispersed mixture of activated oligomers isillustrated in FIG. 7 and described in Examples 32-37 hereinbelow. Stillanother embodiment of a synthesis route for providing a substantiallymonodispersed mixture of activated oligomers is illustrated in FIG. 8and described in Example 38 hereinbelow. Yet another embodiment of asynthesis route for providing a substantially monodispersed mixture ofactivated oligomers is illustrated in FIG. 9 and described in Example 39hereinbelow. Another embodiment of a synthesis route for providing asubstantially monodispersed mixture of activated oligomers isillustrated in FIG. 10 and described in Example 40 hereinbelow.

[0106] The substantially monodispersed mixture of activated oligomersmay be reacted with a substantially monodispersed mixture of insulindrugs under conditions sufficient to provide a mixture of insulindrug-oligomer conjugates. A preferred synthesis is described in Example41 hereinbelow. As will be understood by those skilled in the art, thereaction conditions (e.g., selected molar ratios, solvent mixturesand/or pH) may be controlled such that the mixture of insulindrug-oligomer conjugates resulting from the reaction of thesubstantially monodispersed mixture of activated oligomers and thesubstantially monodispersed mixture of insulin drugs is a substantiallymonodispersed mixture. For example, conjugation at the aminofunctionality of lysine may be suppressed by maintaining the pH of thereaction solution below the pK_(a) of lysine. Alternatively, the mixtureof insulin drug-oligomer conjugates may be separated and isolatedutilizing, for example, HPLC as described below in Example 50 to providea substantially monodispersed mixture of insulin drug-oligomerconjugates, for example mono-, di-, or tri-conjugates. The degree ofconjugation (e.g., whether the isolated molecule is a mono-, di-, ortri-conjugate) of a particular isolated conjugate may be determinedand/or verified utilizing various techniques as will be understood bythose skilled in the art including, but not limited to, massspectroscopy. The particular conjugate structure (e.g., whether theoligomer is at G^(A1), Phe^(B1) or Lys^(B29) a human insulinmonoconjugate) may be determined and/or verified utilizing varioustechniques as will be understood by those skilled in the art including,but not limited to, sequence analysis, peptide mapping, selectiveenzymatic cleavage, and/or endopeptidase cleavage.

[0107] As will be understood by those skilled in the art, one or more ofthe reaction sites on the insulin drug may be blocked by, for example,reacting the insulin drug with a suitable reagent such asN-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl)(N-FMOC). This process may be preferred, for example, when the insulindrug is a polypeptide and it is desired to form an unsaturated conjugate(i.e., a conjugate wherein not all nucleophilic residues are conjugated)having an oligomer at one or more of the N-termini of the polypeptide.Following such blocking, the substantially monodispersed mixture ofblocked insulin drugs may be reacted with the substantiallymonodispersed mixture of activated oligomers to provide a mixture ofinsulin drug-oligomer conjugates having oligomer(s) coupled to one ormore nucleophilic residues and having blocking moieties coupled to othernucleophilic residues. After the conjugation reaction, the insulindrug-oligomer conjugates may be de-blocked as will be understood bythose skilled in the art. If necessary, the mixture of insulindrug-oligomer conjugates may then be separated as described above toprovide a substantially monodispersed mixture of insulin drug-oligomerconjugates. Alternatively, the mixture of insulin drug-oligomerconjugates may be separated prior to de-blocking.

[0108] According to other embodiments of the present invention, asubstantially monodispersed mixture of conjugates is provided where eachconjugate includes human insulin covalently coupled at Lys^(B29) of theinsulin to a carboxylic acid moiety of an oligomer that compriseshexanoic acid covalently coupled at the end distal to the carboxylicacid moiety to a methyl terminated polyethylene glycol moiety having atleast 7 polyethylene glycol subunits. Preferably each conjugate in thesubstantially monodispersed mixture consists of human insulin covalentlycoupled at Lys^(B29) of the insulin to a carboxylic acid moiety of anoligomer that consists of hexanoic acid covalently coupled at the enddistal to the carboxylic acid moiety to a methyl terminated polyethyleneglycol moiety having 7 polyethylene glycol subunits.

[0109] Substantially monodispersed mixtures of conjugates according tothese embodiments of the present invention preferably have improvedproperties when compared with those of polydispersed mixtures. Forexample, a substantially monodispersed mixture of insulin drug-oligomerconjugates preferably has an in vivo activity that is greater than thein vivo activity of a polydispersed mixture of insulin drug-oligomerconjugates having the same number average molecular weight as thesubstantially monodispersed mixture. As will be understood by thoseskilled in the art, the number average molecular weight of thesubstantially monodispersed mixture and the number average weight of thepolydispersed mixture may be measured by various methods including, butnot limited to, size exclusion chromatography such as gel permeationchromatography as described, for example, in H. R. Allcock & F. W.Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).

[0110] As another example, a substantially monodispersed mixture ofinsulin drug-oligomer conjugates preferably has an in vitro activitythat is greater than the in vitro activity of a polydispersed mixture ofinsulin drug-oligomer conjugates having the same number averagemolecular weight as the substantially monodispersed mixture. As will beunderstood by those skilled in the art, the number average molecularweight of the substantially monodispersed mixture and the number averageweight of the polydispersed mixture may be measured by various methodsincluding, but not limited to, size exclusion chromatography.

[0111] The in vitro activity of a particular mixture may be measured byvarious methods, as will be understood by those skilled in the art.Preferably, the in vitro activity is measured using a Cytosensor®Microphysiometer commercially available from Molecular DevicesCorporation of Sunnyvale, Calif. The microphysiometer monitors smallchanges in the rates of extracellular acidification in response to adrug being added to cultured cells in a transwell. This response isproportional to the activity of the molecule under study. Preferably,the in vitro activity of the substantially monodispersed mixture is atleast about 5 percent greater than the in vitro activity of thepolydispersed mixture. More preferably, the in vitro activity of thesubstantially monodispersed mixture is at least about 10 percent greaterthan the in vitro activity of the polydispersed mixture.

[0112] As still another example, a substantially monodispersed mixtureof insulin drug-oligomer conjugates preferably has an increasedresistance to degradation by chymotrypsin when compared to theresistance to degradation by chymotrypsin of a polydispersed mixture ofinsulin drug-oligomer conjugates having the same number averagemolecular weight as the substantially monodispersed mixture. Resistanceto chymotrypsin corresponds to the percent remaining when the moleculeto be tested is digested in chymotrypsin using a procedure similar tothe one outlined in Example 52 below. As will be understood by thoseskilled in the art, the number average molecular weight of thesubstantially monodispersed mixture and the number average weight of thepolydispersed mixture may be measured by various methods including, butnot limited to, size exclusion chromatography. Preferably, theresistance to degradation by chymotrypsin of the substantiallymonodispersed mixture is at least about 10 percent greater than theresistance to degradation by chymotrypsin of the polydispersed mixture.More preferably, the resistance to degradation by chymotrypsin of thesubstantially monodispersed mixture is at least about 20 percent greaterthan the resistance to degradation by chymotrypsin of the polydispersedmixture.

[0113] As yet another example, a substantially monodispersed mixture ofinsulin drug-oligomer conjugates preferably has an inter-subjectvariability that is less than the inter-subject variability of apolydispersed mixture of insulin drug-oligomer conjugates having thesame number average molecular weight as the substantially monodispersedmixture. As will be understood by those skilled in the art, the numberaverage molecular weight of the substantially monodispersed mixture andthe number average weight of the polydispersed mixture may be measuredby various methods including, but not limited to, size exclusionchromatography. The inter-subject variability may be measured by variousmethods as will be understood by those skilled in the art. Theinter-subject variability is preferably calculated as follows. The areaunder a dose response curve (AUC) (i.e., the area between thedose-response curve and a baseline value) is determined for eachsubject. The average AUC for all subjects is determined by summing theAUCs of each subject and dividing the sum by the number of subjects. Theabsolute value of the difference between the subject's AUC and theaverage AUC is then determined for each subject. The absolute values ofthe differences obtained are then summed to give a value that representsthe inter-subject variability. Lower values represent lowerinter-subject variabilities and higher values represent higherinter-subject variabilities. Preferably, the inter-subject variabilityof the substantially monodispersed mixture is at least about 10 percentless than the inter-subject variability of the polydispersed mixture.More preferably, the inter-subject variability of the substantiallymonodispersed mixture is at least about 25 percent less than theinter-subject variability of the polydispersed mixture.

[0114] Substantially monodispersed mixtures of conjugates according toembodiments of the present invention preferably have two or more of theabove-described properties. More preferably, substantially monodispersedmixtures of conjugates according to embodiments of the present inventionhave three or more of the above-described properties. Most preferably,substantially monodispersed mixtures of conjugates according toembodiments of the present invention have all four of theabove-described properties.

[0115] In still other embodiments according to the present invention, amixture of conjugates having a molecular weight distribution with astandard deviation of less than about 22 Daltons is provided. Eachconjugate in the mixture includes an insulin drug coupled to an oligomerthat comprises a polyethylene glycol moiety. The standard deviation ispreferably less than about 14 Daltons and is-more preferably less thanabout 11 Daltons. The molecular weight distribution may be determined bymethods known to those skilled in the art including, but not limited to,size exclusion chromatography such as gel permeation chromatography asdescribed, for example, in H. R. Allcock & F. W. Lampe, CONTEMPORARYPOLYMER CHEMISTRY 394-402 (2d. ed., 1991). The standard deviation of themolecular weight distribution may then be determined by statisticalmethods as will be understood by those skilled in the art.

[0116] The insulin drug is preferably insulin. More preferably, theinsulin drug is human insulin. However, it is to be understood that theinsulin drug may be selected from various insulin drugs known to thoseskilled in the art including, for example, proinsulin, insulin analogs,insulin fragments, and insulin fragment analogs. Insulin analogsinclude, but are not limited to, Asp^(B28) human insulin, Lys^(B28)human insulin, Leu^(B28) human insulin, Val^(B28) human insulin,Ala^(B28) human insulin, Asp^(B28) Pro^(B29) human insulin, Lys^(B28)Pro^(B29) human insulin, Leu^(B28)Pro^(B29) human insulin,Val^(B28)Pro^(B29) human insulin, Ala^(B28)Pro^(B29) human insulin, aswell as analogs provided using the substitution guidelines describedabove. Insulin fragments include, but are not limited to, B22-B30 humaninsulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 humaninsulin, B27-B30 human insulin, B29-B30 human insulin, the A chain ofhuman insulin, and the B chain of human insulin. Insulin fragmentanalogs may be provided by substituting one or more amino acids asdescribed above in an insulin fragment.

[0117] The oligomer may be various oligomers comprising a polyethyleneglycol moiety as will be understood by those skilled in the art.Preferably, the polyethylene glycol moiety of the oligomer has at least2, 3 or 4 polyethylene glycol subunits. More preferably, thepolyethylene glycol moiety has-at least 5 or 6 polyethylene glycolsubunits and, most preferably, the polyethylene glycol moiety has atleast 7 polyethylene glycol subunits.

[0118] The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,additional hydrophilic moieties, lipophilic moieties, spacer moieties,linker moieties, and terminating moieties. The various moieties in theoligomer are covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

[0119] The oligomer may further comprise one or more additionalhydrophilic moieties (i.e., moieties in addition to the polyethyleneglycol moiety) including, but not limited to, sugars, polyalkyleneoxides, and polyamine/PEG copolymers. As polyethylene glycol is apolyalkylene oxide, the additional hydrophilic moiety may be apolyethylene glycol moiety. Adjacent polyethylene glycol moieties willbe considered to be the same moiety if they are coupled by an etherbond. For example, the moiety

—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—

[0120] is a single polyethylene glycol moiety having six polyethyleneglycol subunits. If this moiety were the only hydrophilic moiety in theoligomer, the oligomer would not contain an additional hydrophilicmoiety. Adjacent polyethylene glycol moieties will be considered to bedifferent moieties if they are coupled by a bond other than an etherbond. For example, the moiety

[0121] is a polyethylene glycol moiety having four polyethylene glycolsubunits and an additional hydrophilic moiety having two polyethyleneglycol subunits. Preferably, oligomers according to embodiments of thepresent invention comprise a polyethylene glycol moiety and noadditional hydrophilic moieties.

[0122] The oligomer may further comprise one or more lipophilic moietiesas will be understood by those skilled in the art. The lipophilic moietyis preferably a saturated or unsaturated, linear or branched alkylmoiety or a saturated or unsaturated, linear or branched fatty acidmoiety. When the lipophilic moiety is an alkyl moiety, it is preferablya linear, saturated or unsaturated alkyl moiety having 1 to 28 carbonatoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. Whenthe lipophilic moiety is a fatty acid moiety, it is preferably a naturalfatty acid moiety that is linear, saturated or unsaturated, having 2 to18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5or 6 carbon atoms.

[0123] The oligomer may further comprise one or more spacer moieties aswill be understood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from theinsulin drug, to separate a first hydrophilic or lipophilic moiety froma second hydrophilic or lipophilic moiety, or to separate a hydrophilicmoiety or lipophilic moiety from a linker moiety. Spacer moieties arepreferably selected from the group consisting of sugar, cholesterol andglycerine moieties.

[0124] The oligomer may further comprise one or more linker moietiesthat are used to couple the oligomer with the insulin drug as will beunderstood by those skilled in the art. Linker moieties are preferablyselected from the group consisting of alkyl and fatty acid moieties.

[0125] The oligomer may further comprise one or more terminatingmoieties at the one or more ends of the oligomer which are not coupledto the insulin drug. The terminating moiety is preferably an alkyl oralkoxy moiety, and is more preferably a lower alkyl or lower alkoxymoiety. Most preferably, the terminating moiety is methyl or methoxy.While the terminating moiety is preferably an alkyl or alkoxy moiety, itis to be understood that the terminating moiety may be various moietiesas will be understood by those skilled in the art including, but notlimited to, sugars, cholesterol, alcohols, and fatty acids.

[0126] The oligomer is preferably covalently coupled to the insulindrug. In some embodiments, the insulin drug is coupled to the oligomerutilizing a hydrolyzable bond (e.g., an ester or carbonate bond). Ahydrolyzable coupling may provide an insulin drug-oligomer conjugatethat acts as a prodrug. In certain instances, for example where theinsulin drug-oligomer conjugate is inactive (i.e., the conjugate lacksthe ability to affect the body through the insulin drug's primarymechanism of action), a hydrolyzable coupling may provide for atime-release or controlled-release effect, administering the insulindrug over a given time period as one or more oligomers are cleaved fromtheir respective insulin drug-oligomer conjugates to provide the activedrug. In other embodiments, the insulin drug is coupled to the oligomerutilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or etherbond). Use of a non-hydrolyzable bond may be preferable when it isdesirable to allow the insulin drug-oligomer conjugate to circulate inthe bloodstream for an extended period of time, preferably at least 2hours. When the oligomer is covalently coupled to the insulin drug, theoligomer further comprises one or more bonding moieties that are used tocovalently couple the oligomer with the insulin drug as will beunderstood by those skilled in the art. Bonding moieties are preferablyselected from the group consisting of covalent bond(s), ester moieties,carbonate moieties, carbamate moieties, amide moieties and secondaryamine moieties. More than one moiety on the oligomer may be covalentlycoupled to the insulin drug.

[0127] While the oligomer is preferably covalently coupled to theinsulin drug, it is to be understood that the oligomer may benon-covalently coupled to the insulin drug to form a non-covalentlyconjugated insulin drug-oligomer complex. As will be understood by thoseskilled in the art, non-covalent couplings include, but are not limitedto, hydrogen bonding, ionic bonding, Van der Waals bonding, andmicellular or liposomal encapsulation. According to embodiments of thepresent invention, oligomers may be suitably constructed, modifiedand/or appropriately functionalized to impart the ability fornon-covalent conjugation in a selected manner (e.g., to impart hydrogenbonding capability), as will be understood by those skilled in the art.According to other embodiments of present invention, oligomers may bederivatized with various compounds including, but not limited to, aminoacids, oligopeptides, peptides, bile acids, bile acid derivatives, fattyacids, fatty acid derivatives, salicylic acids, salicylic acidderivatives, aminosalicylic acids, and aminosalicylic acid derivatives.The resulting oligomers can non-covalently couple (complex) with drugmolecules, pharmaceutical products, and/or pharmaceutical excipients.The resulting complexes preferably have balanced lipophilic andhydrophilic properties. According to still other embodiments of thepresent invention, oligomers may be derivatized with amine and/or alkylamines. Under suitable acidic conditions, the resulting oligomers canform non-covalently conjugated complexes with drug molecules,pharmaceutical products and/or pharmaceutical excipients. The productsresulting from such complexation preferably have balanced lipophilic andhydrophilic properties.

[0128] More than one oligomer (i.e., a plurality of oligomers) may becoupled to the insulin drug. The oligomers in the plurality arepreferably the same. However, it is to be understood that the oligomersin the plurality may be different from one another, or, alternatively,some of the oligomers in the plurality may be the same and some may bedifferent. When a plurality of oligomers are coupled to the insulindrug, it may be preferable to couple one or more of the oligomers to theinsulin drug with hydrolyzable bonds and couple one or more of theoligomers to the insulin drug with non-hydrolyzable bonds.Alternatively, all of the bonds coupling the plurality of oligomers tothe insulin drug may be. hydrolyzable, but have varying degrees ofhydrolyzability such that, for example, one or more of the oligomers israpidly removed from the insulin drug by hydrolysis in the body and oneor more of the oligomers is slowly removed from the insulin drug byhydrolysis in the body.

[0129] The oligomer may be coupled to the insulin drug at variousnucleophilic residues of the insulin drug including, but not limited to,nucleophilic hydroxyl functions and/or amino functions. When the insulindrug is a polypeptide, a nucleophilic hydroxyl function may be found,for example, at serine and/or tyrosine residues, and a nucleophilicamino function may be found, for example, at histidine and/or lysineresidues, and/or at the one or more N-termini of the polypeptide. Whenan oligomer is coupled to the one or more N-termini of the insulinpolypeptide, the coupling preferably forms a secondary amine. When theinsulin drug is human insulin, for example, the oligomer may be coupledto an amino functionality of the insulin, including the aminofunctionality of Gly^(A1), the amino functionality of Phe^(B1), and theamino functionality of Lys^(B29). When one oligomer is coupled to thehuman insulin, the oligomer is preferably coupled to the aminofunctionality of Lys^(B29). When two oligomers are coupled to the humaninsulin, the oligomers are preferably coupled to the amino functionalityof Phe^(B1) and the amino functionality of LyS^(B29). While more thanone oligomer may be coupled to the human insulin, a higher activity(improved glucose lowering ability) is observed for the mono-conjugatedhuman insulin.

[0130] Mixtures of insulin drug-oligomer conjugates having a molecularweight distribution with a standard deviation of less than about 22Daltons may be synthesized by various methods. For example, a mixture ofoligomers having a molecular weight distribution with a standarddeviation of less than about 22 Daltons consisting of carboxylic acidand polyethylene glycol is synthesized by contacting a mixture ofcarboxylic acid having a molecular weight distribution with a standarddeviation of less than about 22 Daltons with a mixture of polyethyleneglycol having a molecular weight distribution with a standard deviationof less than about 22 Daltons under conditions sufficient to provide amixture of oligomers having a molecular weight distribution with astandard deviation of less than about 22 Daltons. The oligomers of themixture having a molecular weight distribution with a standard deviationof less than about 22 Daltons are then activated so that they arecapable of reacting with an insulin drug to provide an insulindrug-oligomer conjugate. One embodiment of a synthesis route forproviding a mixture of activated oligomers having a molecular weightdistribution with a standard deviation of less than about 22 Daltons isillustrated in FIG. 3 and described in Examples 11-18 hereinbelow.Another embodiment of a synthesis route for providing a mixture ofactivated oligomers having a molecular weight distribution with astandard deviation of less than about 22 Daltons is illustrated in FIG.4 and described in Examples 19-24 hereinbelow. Still another embodimentof a synthesis route for providing a mixture of activated oligomershaving a molecular weight distribution with a standard deviation of lessthan about 22 Daltons is illustrated in FIG. 5 and described in Examples25-29 hereinbelow. Yet another embodiment of a synthesis route forproviding a mixture of activated oligomers having a molecular weightdistribution with a standard deviation of less than about 22 Daltons isillustrated in FIG. 6 and described in Examples 30-31 hereinbelow.Another embodiment of a synthesis route for providing a mixture ofactivated oligomers having a molecular weight distribution with astandard deviation of less than about 22 Daltons is illustrated in FIG.7 and described in Examples 32-37 hereinbelow. Still another embodimentof a synthesis route for providing a mixture of activated oligomershaving a molecular weight distribution with a standard deviation of lessthan about 22 Daltons is illustrated in FIG. 8 and described in Example38 hereinbelow. Yet another embodiment of a synthesis route forproviding a mixture of activated oligomers having a molecular weightdistribution with a standard deviation of less than about 22 Daltons isillustrated in FIG. 9 and described in Example 39 hereinbelow. Anotherembodiment of a synthesis route for providing a mixture of activatedoligomers having a molecular weight distribution with a standarddeviation of less than about 22 Daltons is illustrated in FIG. 10 anddescribed in Example 40 hereinbelow.

[0131] The mixture of activated oligomers having a molecular weightdistribution with a standard deviation of less than about 22 Daltons isreacted with a mixture of insulin drugs having a molecular weightdistribution with a standard deviation of less than about 22 Daltonsunder conditions sufficient to provide a mixture of insulindrug-oligomer conjugates. A preferred synthesis is described in Example41 hereinbelow. As will be understood by those skilled in the art, thereaction conditions (e.g., selected molar ratios, solvent mixturesand/or pH) may be controlled such that the mixture of insulindrug-oligomer conjugates resulting from the reaction of the mixture ofactivated oligomers having a molecular weight distribution with astandard deviation of less than about 22 Daltons and the mixture ofinsulin drugs having a molecular weight distribution with a standarddeviation of less than about 22 Daltons is a mixture having a molecularweight distribution with a standard deviation of less than about 22Daltons. For example, conjugation at the amino functionality of lysinemay be suppressed by maintaining the pH of the reaction solution belowthe pK_(a) of lysine. Alternatively, the mixture of insulindrug-oligomer conjugates may be separated and isolated utilizing, forexample, HPLC as described below in Example 50 to provide a mixture ofinsulin drug-oligomer conjugates, for example mono-, di-, ortri-conjugates, having a molecular weight distribution with a standarddeviation of less than about 22 Daltons. The degree of conjugation(e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate)of a particular isolated conjugate may be determined and/or verifiedutilizing various techniques as will be understood by those skilled inthe art including, but not limited to, mass spectroscopy. The particularconjugate structure (e.g., whether the oligomer is at Gly^(A1), Phe^(B1)or Lys^(B29) of a human insulin monoconjugate) may be determined and/orverified utilizing various techniques as will be understood by thoseskilled in the art including, but not limited to, sequence analysis,peptide mapping, selective enzymatic cleavage, and/or endopeptidasecleavage.

[0132] As will be understood by those skilled in the art, one or more ofthe reaction sites on the insulin drug may be blocked by, for example,reacting the insulin drug with a suitable blocking reagent such asN-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl)(N-FMOC). This process may be preferred, for example, when the insulindrug is a polypeptide and it is desired to form an unsaturated conjugate(i.e., a conjugate wherein not all nucleophilic residues are conjugated)having an oligomer at the N-terminus of the polypeptide. Following suchblocking, the mixture of blocked insulin drugs having a molecular weightdistribution with a standard deviation of less than about 22 Daltons maybe reacted with the mixture of activated oligomers having a molecularweight distribution with a standard deviation of less than about 22Daltons to provide a mixture of insulin drug-oligomer conjugates havingoligomer(s) coupled to one or more nucleophilic residues and havingblocking moieties coupled to other nucleophilic residues. After theconjugation reaction, the insulin drug-oligomer conjugates may bede-blocked as will be understood by those skilled in the art. Ifnecessary, the mixture of insulin drug-oligomer conjugates may then beseparated as described above to provide a mixture of insulindrug-oligomer conjugates having a molecular weight distribution with astandard deviation of less than about 22 Daltons. Alternatively, themixture of insulin drug-oligomer conjugates may be separated prior tode-blocking.

[0133] Mixtures of insulin drug-oligomer conjugates having a molecularweight distribution with a standard deviation of less than about 22Daltons according to these embodiments of the present inventionpreferably have improved properties when compared with those ofpolydispersed mixtures. For example, a mixture of insulin drug-oligomerconjugates having a molecular weight distribution with a standarddeviation of less than about 22 Daltons preferably has an in vivoactivity that is greater than the in vivo activity of a polydispersedmixture of insulin drug-oligomer conjugates having the same numberaverage molecular weight as the mixture of insulin drug-oligomerconjugates having a molecular weight distribution with a standarddeviation of less than about 22 Daltons. As will be understood by thoseskilled in the art, the number average molecular weight of the mixtureof insulin drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltons andthe number average weight of the polydispersed mixture may be measuredby various methods including, but not limited to, size exclusionchromatography such as gel permeation chromatography as described, forexample, in H. R. Allcock & F. W. Lampe, CONTEMPORARY POLYMER CHEMISTRY394-402 (2d. ed., 1991).

[0134] As another example, a mixture of insulin drug-oligomer conjugateshaving a molecular weight distribution with a standard deviation of lessthan about 22 Daltons preferably has an in vitro activity that isgreater than the in vitro activity of a polydispersed mixture of insulindrug-oligomer conjugates having the same number average molecular weightas the mixture of insulin drug-oligomer conjugates having a molecularweight distribution with a standard deviation of less than about 22Daltons. As will be understood by those skilled in the art, the numberaverage molecular weight of the mixture of insulin drug-oligomerconjugates having a molecular weight distribution with a standarddeviation of less than about 22 Daltons and the number average weight ofthe polydispersed mixture may be measured by various methods including,but not limited to, size exclusion chromatography.

[0135] The in vitro activity of a particular mixture may be measured byvarious methods, as will be understood by those skilled in the art.Preferably, the in vitro activity is measured using a Cytosensor®Microphysiometer commercially available from Molecular DevicesCorporation of Sunnyvale, Calif. The microphysiometer monitors smallchanges in the rates of extracellular acidification in response to adrug being added to cultured cells in a transwell. This response isproportional to the activity of the molecule under study. Preferably,the in vitro activity of the mixture of insulin drug-oligomer conjugateshaving a molecular weight distribution with a standard deviation of lessthan about 22 Daltons is at least about 5 percent greater than the invitro activity of the polydispersed mixture. More preferably, the invitro activity of the mixture of insulin drug-oligomer conjugates havinga molecular weight distribution with a standard deviation of less thanabout 22 Daltons is at least about 10 percent greater than the in vitroactivity of the polydispersed mixture.

[0136] As still another example, a mixture of insulin drug-oligomerconjugates having a molecular weight distribution with a standarddeviation of less than about 22 Daltons preferably has an increasedresistance to degradation by chymotrypsin when compared to theresistance to degradation by chymotrypsin of a polydispersed mixture ofinsulin drug-oligomer conjugates having the same number averagemolecular weight as the mixture of insulin drug-oligomer conjugateshaving a molecular weight distribution with a standard deviation of lessthan about 22 Daltons. Resistance to chymotrypsin corresponds to thepercent remaining when the molecule to be tested is digested inchymotrypsin using a procedure similar to the one outlined in Example 52below. As will be understood by those skilled in the art, the numberaverage molecular weight of the mixture of insulin drug-oligomerconjugates having a molecular weight distribution with a standarddeviation of less than about 22 Daltons and the number average weight ofthe polydispersed mixture may be measured by various methods including,but not limited to, size exclusion chromatography. Preferably, theresistance to degradation by chymotrypsin of the mixture of insulindrug-oligomer conjugates having a molecular weight distribution with astandard deviation of less than about 22 Daltons is at least about 10percent greater than the resistance to degradation by chymotrypsin ofthe polydispersed mixture. More preferably, the resistance todegradation by chymotrypsin of the mixture of insulin drug-oligomerconjugates having a molecular weight distribution with a standarddeviation of less than about 22 Daltons is at least about 20 percentgreater than the resistance to degradation by chymotrypsin of thepolydispersed mixture.

[0137] As yet another example, a mixture of insulin drug-oligomerconjugates having a molecular weight distribution with a standarddeviation of less than about 22 Daltons preferably has an inter-subjectvariability that is less than the inter-subject variability of apolydispersed mixture of insulin drug-oligomer conjugates having thesame number average molecular weight as the mixture of insulindrug-oligomer conjugates having a molecular weight distribution with astandard deviation of less than about 22 Daltons. As will be understoodby those skilled in the art, the number average molecular weight of themixture of insulin drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltons andthe number average weight of the polydispersed mixture may be measuredby various methods including, but not limited to, size exclusionchromatography. The inter-subject variability may be measured by variousmethods as will be understood by those skilled in the art. Theinter-subject variability is preferably calculated as follows. The areaunder a dose response curve (AUC) (i.e., the area between thedose-response curve and a baseline value) is determined for eachsubject. The average AUC for all subjects is determined by summing theAUCs of each subject and dividing the sum by the number of subjects. Theabsolute value of the difference between the subject's AUC and theaverage AUC is then determined for each subject. The absolute values ofthe differences obtained are then summed to give a value that representsthe inter-subject variability. Lower values represent lowerinter-subject variabilities and higher values represent higherinter-subject variabilities. Preferably, the inter-subject variabilityof the mixture of insulin drug-oligomer conjugates having a molecularweight distribution with a standard deviation of less than about 22Daltons is at least about 10 percent less than the inter-subjectvariability of the polydispersed mixture. More preferably, theinter-subject variability of the mixture of insulin drug-oligomerconjugates having a molecular weight distribution with a standarddeviation of less than about 22 Daltons is at least about 25 percentless than the inter-subject variability of the polydispersed mixture.

[0138] Mixtures of insulin drug-oligomer conjugates having a molecularweight distribution with a standard deviation of less than about 22Daltons according to embodiments of the present invention preferablyhave two or more of the above-described properties. More preferably,mixtures of insulin drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltonsaccording to embodiments of the present invention have three or more ofthe above-described properties. Most preferably, mixtures of insulindrug-oligomer conjugates having a molecular weight distribution with astandard deviation of less than about 22 Daltons according toembodiments of the present invention have all four of theabove-described properties.

[0139] According to yet other embodiments of the present invention, amixture of conjugates is provided where each conjugate includes aninsulin drug coupled to an oligomer that comprises a polyethylene glycolmoiety, and the mixture has a dispersity coefficient (DC) greater than10,000 where${D\quad C} = \frac{\left( {\sum\limits_{i = 1}^{n}{N_{i}M_{i}}} \right)^{2}}{{\sum\limits_{i = 1}^{n}{N_{i}M_{i}^{2}{\sum\limits_{i = 1}^{n}N_{i}}}} - \left( {\sum\limits_{i = 1}^{n}{N_{i}M_{i}}} \right)^{2}}$

[0140] wherein:

[0141] n is the number of different molecules in the sample;

[0142] N_(i) is the number of i^(th) molecules in the sample; and

[0143] M_(i) is the mass of the i^(th) molecule.

[0144] The mixture of conjugates preferably has a dispersity coefficientgreater than 100,000. More preferably, the dispersity coefficient of theconjugate mixture is greater than 500,000 and, most preferably, thedispersity coefficient is greater than 10,000,000. The variables n,N_(i), and M_(i) may be determined by various methods as will beunderstood by those skilled in the art, including, but not limited to,methods described below in Example 49.

[0145] The insulin drug is preferably insulin. More preferably, theinsulin drug is human insulin. However, it is to be understood that theinsulin drug may be selected from various insulin drugs known to thoseskilled in the art including, for example, proinsulin, insulin analogs,insulin fragments, and insulin fragment analogs. Insulin analogsinclude, but are not limited to, Asp^(B28) human insulin, Lys^(B28)human insulin, Leu^(B28) human insulin, Val^(B28) human insulin,Ala^(B28) human insulin, Asp^(B28)Pro^(B29) human insulin,Lys^(B28)Pro^(B29) human insulin, Leu^(B28)Pro^(B29) human insulin,Val^(B28)Pro^(B29) human insulin, Ala^(B28)Pro^(B29) human insulin, aswell as analogs provided using the substitution guidelines describedabove. Insulin fragments include, but are not limited to, B22-B30 humaninsulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 humaninsulin, B27-B30 human insulin, B29-B30 human insulin, the A chain ofhuman insulin, and the B chain of human insulin. Insulin fragmentanalogs may be provided by substituting one or more amino acids asdescribed above in an insulin fragment.

[0146] The oligomer may be various oligomers comprising a polyethyleneglycol moiety as will be understood by those skilled in the art.Preferably, the polyethylene glycol moiety of the oligomer has at least2, 3 or 4 polyethylene glycol subunits. More preferably, thepolyethylene glycol moiety has at least 5 or 6 polyethylene glycolsubunits and, most preferably, the polyethylene glycol moiety has atleast 7 polyethylene glycol subunits.

[0147] The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,additional hydrophilic moieties, lipophilic moieties, spacer moieties,linker moieties, and terminating moieties. The various moieties in theoligomer are covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

[0148] The oligomer may further comprise one or more additionalhydrophilic moieties (i.e., moieties in addition to the polyethyleneglycol moiety) including, but not limited to, sugars, polyalkyleneoxides, and polyamine/PEG copolymers. As polyethylene glycol is apolyalkylene oxide, the additional hydrophilic moiety may be apolyethylene glycol moiety. Adjacent polyethylene glycol moieties willbe considered to be the same moiety if they are coupled by an etherbond. For example, the moiety

—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—

[0149] is a single polyethylene glycol moiety having six polyethyleneglycol subunits. If this moiety were the only hydrophilic moiety in theoligomer, the oligomer would hot contain an additional hydrophilicmoiety. Adjacent polyethylene glycol moieties will be considered to bedifferent moieties if they are coupled by a bond other than an etherbond. For example, the moiety

[0150] is a polyethylene glycol moiety having four polyethylene glycolsubunits and an additional hydrophilic moiety having two polyethyleneglycol subunits. Preferably, oligomers according to embodiments of thepresent invention comprise a polyethylene glycol moiety and noadditional hydrophilic moieties.

[0151] The oligomer may further comprise one or more lipophilic moietiesas will be understood by those skilled in the art. The lipophilic moietyis preferably a saturated or unsaturated, linear or branched alkylmoiety or a saturated or unsaturated, linear or branched fatty acidmoiety. When the lipophilic moiety is an alkyl moiety, it is preferablya linear, saturated or unsaturated alkyl moiety having 1 to 28 carbonatoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. Whenthe lipophilic moiety is a fatty acid moiety, it is preferably a naturalfatty acid moiety that is linear, saturated or unsaturated, having 2 to18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5or 6 carbon atoms.

[0152] The oligomer may further comprise one or more spacer moieties aswill be understood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from theinsulin drug, to separate a first hydrophilic or lipophilic moiety froma second hydrophilic or lipophilic moiety, or to separate a hydrophilicmoiety or lipophilic moiety from a linker moiety. Spacer moieties arepreferably selected from the group consisting of sugar, cholesterol andglycerine moieties.

[0153] The oligomer may further comprise one or more linker moietiesthat are used to couple the oligomer with the insulin drug as will beunderstood by those skilled in the art. Linker moieties are preferablyselected from the group consisting of alkyl and fatty acid moieties.

[0154] The oligomer may further comprise one or more terminatingmoieties at the one or more ends of the oligomer which are not coupledto the insulin drug. The terminating moiety is preferably an alkyl oralkoxy moiety, and is more preferably a lower alkyl or lower alkoxymoiety. Most preferably, the terminating moiety is methyl or methoxy.While the terminating moiety is preferably an alkyl or alkoxy moiety, itis to be understood that the terminating moiety may be various moietiesas will be understood by those skilled in the art including, but notlimited to, sugars, cholesterol, alcohols, and fatty acids.

[0155] The oligomer is preferably covalently coupled to the insulindrug. In some embodiments, the insulin drug is coupled to the oligomerutilizing a hydrolyzable bond (e.g., an ester or carbonate bond). Ahydrolyzable coupling may provide an insulin drug-oligomer conjugatethat acts as a prodrug. In certain instances, for example where theinsulin drug-oligomer conjugate is inactive (i.e., the conjugate lacksthe ability to affect the body through the insulin drug's primarymechanism of action), a hydrolyzable coupling may provide for atime-release or controlled-release effect, administering the insulindrug over a given time period as one or more oligomers are cleaved fromtheir respective insulin drug-oligomer conjugates to provide the activedrug. In other embodiments, the insulin drug is coupled to the oligomerutilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or etherbond). Use of a non-hydrolyzable bond may be preferable when it isdesirable to allow the insulin drug-oligomer conjugate to circulate inthe bloodstream for an extended period of time, preferably at least 2hours. When the oligomer is covalently coupled to the insulin drug, theoligomer further comprises one or more bonding moieties that are used tocovalently couple the oligomer with the insulin drug as will beunderstood by those skilled in the art. Bonding moieties are preferablyselected from the group consisting of covalent bond(s), ester moieties,carbonate moieties, carbamate moieties, amide moieties and secondaryamine moieties. More than one moiety on the oligomer may be covalentlycoupled to the insulin drug.

[0156] While the oligomer is preferably covalently coupled to theinsulin drug, it is to be understood that the oligomer may benon-covalently coupled to the insulin drug to form a non-covalentlyconjugated insulin drug-oligomer complex. As will be understood by thoseskilled in the art, non-covalent couplings include, but are not limitedto, hydrogen bonding, ionic bonding, Van der Waals bonding, andmicellular or liposomal encapsulation. According to embodiments of thepresent invention, oligomers may be suitably constructed, modifiedand/or appropriately functionalized to impart the ability fornon-covalent conjugation in a selected manner (e.g., to impart hydrogenbonding capability), as will be understood by those skilled in the art.According to other embodiments of present invention, oligomers may bederivatized with various compounds including, but not limited to, aminoacids, oligopeptides, peptides, bile acids, bile acid derivatives, fattyacids, fatty acid derivatives, salicylic acids, salicylic acidderivatives, aminosalicylic acids, and aminosalicylic acid derivatives.The resulting oligomers can non-covalently couple (complex) with drugmolecules, pharmaceutical products, and/or pharmaceutical excipients.The resulting complexes preferably have balanced lipophilic andhydrophilic properties. According to still other embodiments of thepresent invention, oligomers may be derivatized with amine and/or alkylamines. Under suitable acidic conditions, the resulting oligomers canform non-covalently conjugated complexes with drug molecules,pharmaceutical products and/or pharmaceutical excipients. The productsresulting from such complexation preferably have balanced lipophilic andhydrophilic properties.

[0157] More than one oligomer (i.e., a plurality of oligomers) may becoupled to the insulin drug. The oligomers in the plurality arepreferably the same. However, it is to be understood that the oligomersin the plurality may be different from one another, or, alternatively,some of the oligomers in the plurality may be the same and some may bedifferent. When a plurality of oligomers are coupled to the insulindrug, it may be preferable to couple one or more of the oligomers to theinsulin drug with hydrolyzable bonds and couple one or more of theoligomers to the insulin drug with non-hydrolyzable bonds.Alternatively, all of the bonds coupling the plurality of oligomers tothe insulin drug may be hydrolyzable, but have varying degrees ofhydrolyzability such that, for example, one or more of the oligomers israpidly removed from the insulin drug by hydrolysis in the body and oneor more of the oligomers is slowly removed from the insulin drug byhydrolysis in the body.

[0158] The oligomer may be coupled to the insulin drug at variousnucleophilic residues of the insulin drug including, but not limited to,nucleophilic hydroxyl functions and/or amino functions. When the insulindrug is a polypeptide, a nucleophilic hydroxyl function may be found,for example, at serine and/or tyrosine residues, and a nucleophilicamino function may be found, for example, at histidine and/or lysineresidues, and/or at the one or more N-termini of the polypeptide. Whenan oligomer is coupled to the one or more N-termini of the insulinpolypeptide, the coupling preferably forms a secondary amine. When theinsulin drug is human insulin, for example, the oligomer may be coupledto an amino functionality of the insulin, including the aminofunctionality of Gly^(A1), the amino functionality of Phe^(B1), and theamino functionality of Lys^(B29) When one oligomer is coupled to thehuman insulin, the oligomer is preferably coupled to the aminofunctionality of Lys^(B29) When two oligomers are coupled to the humaninsulin, the oligomers are preferably coupled to the amino functionalityof Phe^(B1) and the amino functionality of Lys^(B29). While more thanone oligomer may be coupled to the human insulin, a higher activity(improved glucose lowering ability) is observed for the mono-conjugatedhuman insulin.

[0159] Mixtures of insulin drug-oligomer conjugates having a dispersitycoefficient greater than 10,000 may be synthesized by various methods.For example, a mixture of oligomers having a dispersity coefficientgreater than 10,000 consisting of carboxylic acid and polyethyleneglycol is synthesized by contacting a mixture of carboxylic acid havinga dispersity coefficient greater than 10,000 with a mixture ofpolyethylene glycol having a dispersity coefficient greater than 10,000under conditions sufficient to provide a mixture of oligomers having adispersity coefficient greater than 10,000. The oligomers of the mixturehaving a dispersity coefficient greater than 10,000 are then activatedso that they are capable of reacting with an insulin drug to provide aninsulin drug-oligomer conjugate. One embodiment of a synthesis route forproviding a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 3 and describedin Examples 11-18 hereinbelow. Another embodiment of a synthesis routefor providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 4 and describedin Examples 19-24 hereinbelow. Still another embodiment of a synthesisroute for providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 5 and describedin Examples 25-29 hereinbelow. Yet another embodiment of a synthesisroute for providing a mixture of activated oligomers having a dispersitycoefficient greater than 10.000 is illustrated in FIG. 6 and describedin Examples 30-31 hereinbelow. Another embodiment of a synthesis routefor providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 7 and describedin Examples 32-37 hereinbelow. Still another embodiment of a synthesisroute for providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 8 and describedin Example 38 hereinbelow. Yet another embodiment of a synthesis routefor providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 9 and describedin Example 39 hereinbelow. Another embodiment of a synthesis route forproviding a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 10 and describedin Example 40 hereinbelow.

[0160] The mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is reacted with a mixture of insulindrugs having a dispersity coefficient greater than 10,000 underconditions sufficient to provide a mixture of insulin drug-oligomerconjugates. A preferred synthesis is described in Example 41hereinbelow. As will be understood by those skilled in the art, thereaction conditions (e.g., selected molar ratios, solvent mixturesand/or pH) may be controlled such that the mixture of insulindrug-oligomer conjugates resulting from the reaction of the mixture ofactivated oligomers having a dispersity coefficient greater than 10,000and the mixture of insulin drugs having a dispersity coefficient greaterthan 10,000 is a mixture having a dispersity coefficient greater than10,000. For example, conjugation at the amino functionality of lysinemay be suppressed by maintaining the pH of the reaction solution belowthe pK_(a) of lysine. Alternatively, the mixture of insulindrug-oligomer conjugates may be separated and isolated utilizing, forexample, HPLC as described below in Example 50 to provide a mixture ofinsulin drug-oligomer conjugates, for example mono-, di-, ortri-conjugates, having a dispersity coefficient greater than 10,000. Thedegree of conjugation (e.g., whether the isolated molecule is a mono-,di-, or tri-conjugate) of a particular isolated conjugate may bedetermined and/or verified utilizing various techniques as will beunderstood by those skilled in the art including, but not limited to,mass spectroscopy. The particular conjugate structure (e.g., whether theoligomer is at Gly^(A1), Phe^(B1) or Lys^(B29) of a human insulinmonoconjugate) may be determined and/or verified utilizing varioustechniques as will be understood by those skilled in the art including,but not limited to, sequence analysis, peptide mapping, selectiveenzymatic cleavage, and/or endopeptidase cleavage.

[0161] As will be understood by those skilled in the art, one or more ofthe reaction sites on the insulin drug may be blocked by, for example,reacting the insulin drug with a suitable blocking reagent such asN-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl)(N-FMOC). This process may be preferred, for example, when the insulindrug is a polypeptide and it is desired to form an unsaturated conjugate(i.e., a conjugate wherein not all nucleophilic residues are conjugated)having an oligomer at the one or more N-termini of the polypeptide.Following such blocking, the mixture of blocked insulin drugs having adispersity coefficient greater than 10,000 may be reacted with themixture of activated oligomers having a dispersity coefficient greaterthan 10,000 to provide a mixture of insulin drug-oligomer conjugateshaving oligomer(s) coupled to one or more nucleophilic residues andhaving blocking moieties coupled to other nucleophilic residues. Afterthe conjugation reaction, the insulin drug-oligomer conjugates may bede-blocked as will be understood by those skilled in the art. Ifnecessary, the mixture of insulin drug-oligomer conjugates may then beseparated as described above to provide a mixture of insulindrug-oligomer conjugates having a dispersity coefficient greater than10,000. Alternatively, the mixture of insulin drug-oligomer conjugatesmay be separated prior to de-blocking.

[0162] Mixtures of insulin drug-oligomer conjugates having a dispersitycoefficient greater than 10,000 according to these embodiments of thepresent invention preferably have improved properties when compared withthose of polydispersed mixtures. For example, a mixture of insulindrug-oligomer conjugates having a dispersity coefficient greater than10,000 preferably has an in vivo activity that is greater than the invivo activity of a polydispersed mixture of insulin drug-oligomerconjugates having the same number average molecular weight as themixture of insulin drug-oligomer conjugates having a dispersitycoefficient greater than 10,000. As will be understood by those skilledin the art, the number average molecular weight of the mixture ofinsulin drug-oligomer conjugates having a dispersity coefficient greaterthan 10,000 and the number average weight of the polydispersed mixturemay be measured by various methods including, but not limited to, sizeexclusion chromatography such as gel permeation chromatography asdescribed, for example, in H. R. Allcock & F. W. Lampe, CONTEMPORARYPOLYMER CHEMISTRY 394-402 (2d. ed., 1991).

[0163] As another example, a mixture of insulin drug-oligomer conjugateshaving a dispersity coefficient greater than 10,000 preferably has an invitro activity that is greater than the in vitro activity of apolydispersed mixture of insulin drug-oligomer conjugates having thesame number average molecular weight as the mixture of insulindrug-oligomer conjugates having a dispersity coefficient greater than10,000. As will be understood by those skilled in the art, the numberaverage molecular weight of the mixture of insulin drug-oligomerconjugates having a dispersity coefficient greater than 10,000 and thenumber average weight of the polydispersed mixture may be measured byvarious methods including, but not limited to, size exclusionchromatography.

[0164] The in vitro activity of a particular mixture may be measured byvarious methods, as will be understood by those skilled in the art.Preferably, the in vitro activity is measured using a Cytosensor®Microphysiometer commercially available from Molecular DevicesCorporation of Sunnyvale, Calif. The microphysiometer monitors smallchanges in the rates of extracellular acidification in response to adrug being added to cultured cells in a transwell. This response isproportional to the activity of the molecule under study. Preferably,the in vitro activity of the mixture of insulin drug-oligomer conjugateshaving a dispersity coefficient greater than 10,000 is at least about 5percent greater than the in vitro activity of the polydispersed mixture.More preferably, the in vitro activity of the mixture of insulindrug-oligomer conjugates having a dispersity coefficient greater than10,000 is at least about 10 percent greater than the in vitro activityof the polydispersed mixture.

[0165] As still another example, a mixture of insulin drug-oligomerconjugates having a dispersity coefficient greater than 10,000preferably has an increased resistance to degradation by chymotrypsinwhen compared to the resistance to degradation by chymotrypsin of apolydispersed mixture of insulin drug-oligomer conjugates having thesame number average molecular weight as the mixture of insulindrug-oligomer conjugates having a dispersity coefficient greater than10,000. Resistance to chymotrypsin corresponds to the percent remainingwhen the molecule to be tested is digested in chymotrypsin using aprocedure similar to the one outlined in Example 52 below. As will beunderstood by those skilled in the art, the number average molecularweight of the mixture of insulin drug-oligomer conjugates having adispersity coefficient greater than 10,000 and the number average weightof the polydispersed mixture may be measured by various methodsincluding, but not limited to, size exclusion chromatography.Preferably, the resistance to degradation by chymotrypsin of the mixtureof insulin drug-oligomer conjugates having a dispersity coefficientgreater than 10,000 is at least about 10 percent greater than theresistance to degradation by chymotrypsin of the polydispersed mixture.More preferably, the resistance to degradation by chymotrypsin of themixture of insulin drug-oligomer conjugates having a dispersitycoefficient greater than 10,000 is at least about 20 percent greaterthan the resistance to degradation by chymotrypsin of the polydispersedmixture.

[0166] As yet another example, a mixture of insulin drug-oligomerconjugates having a dispersity coefficient greater than 10,000preferably has an inter-subject variability that is less than theinter-subject variability of a polydispersed mixture of insulindrug-oligomer conjugates having the same number average molecular weightas the mixture of insulin drug-oligomer conjugates having a dispersitycoefficient greater than 10,000. As will be understood by those skilledin the art, the number average molecular weight of the mixture ofinsulin drug-oligomer conjugates having a dispersity coefficient greaterthan 10,000 and the number average weight of the polydispersed mixturemay be measured by various methods including, but not limited to, sizeexclusion chromatography. The inter-subject variability may be measuredby various methods as will be understood by those skilled in the art.The inter-subject variability is preferably calculated as follows. Thearea under a dose response curve (AUC) (i.e., the area between thedose-response curve and a baseline value) is determined for eachsubject. The average AUC for all subjects is determined by summing theAUCs of each subject and dividing the sum by the number of subjects. Theabsolute value of the difference between the subject's AUC and theaverage AUC is then determined for each subject. The absolute values ofthe differences obtained are then summed to give a value that representsthe inter-subject variability. Lower values represent lowerinter-subject variabilities and higher values represent higherinter-subject variabilities. Preferably, the inter-subject variabilityof the mixture of insulin drug-oligomer conjugates having a dispersitycoefficient greater than 10,000 is at least about 10 percent less thanthe inter-subject variability of the polydispersed mixture. Morepreferably, the inter-subject variability of the mixture of insulindrug-oligomer conjugates having a dispersity coefficient greater than10,000 is at least about 25 percent less than the inter-subjectvariability of the polydispersed mixture.

[0167] Mixtures of insulin drug-oligomer conjugates having a dispersitycoefficient greater than 10,000 according to embodiments of the presentinvention preferably have two or more of the above-described properties.More preferably, mixtures of insulin drug-oligomer conjugates having adispersity coefficient greater than 10,000 according to embodiments ofthe present invention have three or more of the above-describedproperties. Most preferably, mixtures of insulin drug-oligomerconjugates having a dispersity coefficient greater than 10,000 accordingto embodiments of the present invention have all four of theabove-described properties.

[0168] According to other embodiments of the present invention, amixture of conjugates in which each conjugate includes an insulin drugcoupled to an oligomer, and has the same number of polyethylene glycolsubunits is provided.

[0169] The insulin drug is preferably insulin. More preferably, theinsulin drug is human insulin. However, it is to be understood that theinsulin drug may be selected from various insulin drugs known to thoseskilled in the art including, for example, proinsulin, insulin analogs,insulin fragments, and insulin fragment analogs. Insulin analogsinclude, but are not limited to, ASP^(B28) human insulin, Lys^(B28)human insulin, Leu^(B28) human insulin, Val^(B28) human insulin,Ala^(B28) human insulin, Asp^(B28)Pro^(B29) human insulin, LyS^(B28)Pro^(B29) human insulin, Leu^(B28)Pro^(B29) human insulin,Val^(B28)Pro^(B29) human insulin, Ala^(B28)Pro^(B29) human insulin, aswell as analogs provided using the substitution guidelines describedabove. Insulin fragments include, but are not limited to, B22-B30 humaninsulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 humaninsulin, B27-B30 human insulin, B29-B30 human insulin, the A chain ofhuman insulin, and the B chain of human insulin. Insulin fragmentanalogs may be provided by substituting one or more amino acids asdescribed above in an insulin fragment.

[0170] The oligomer may be various oligomers comprising a polyethyleneglycol moiety as will be understood by those skilled in the art.Preferably, the polyethylene glycol moiety of the oligomer has at least2, 3 or 4 polyethylene glycol subunits. More preferably, thepolyethylene glycol moiety has at least 5 or 6 polyethylene glycolsubunits and, most preferably, the polyethylene glycol moiety has atleast 7 polyethylene glycol subunits.

[0171] The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,additional hydrophilic moieties, lipophilic moieties, spacer moieties,linker moieties, and terminating moieties. The various moieties in theoligomer are covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

[0172] The oligomer may further comprise one or more additionalhydrophilic moieties (i.e., moieties in addition to the polyethyleneglycol moiety) including, but not limited to, sugars, polyalkyleneoxides, and polyamine/PEG copolymers. As polyethylene glycol is apolyalkylene oxide, the additional hydrophilic moiety may be apolyethylene glycol moiety. Adjacent polyethylene glycol moieties willbe considered to be the same moiety if they are coupled by an etherbond. For example, the moiety

—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—

[0173] is a single polyethylene glycol moiety having six polyethyleneglycol subunits. If this moiety were the only hydrophilic moiety in theoligomer, the oligomer would not contain an additional hydrophilicmoiety. Adjacent polyethylene glycol moieties will be considered to bedifferent moieties if they are coupled by a bond other than an etherbond. For example, the moiety

[0174] is a polyethylene glycol moiety having four polyethylene glycolsubunits and an additional hydrophilic moiety having two polyethyleneglycol subunits. Preferably, oligomers according to embodiments of thepresent invention comprise a polyethylene glycol moiety and noadditional hydrophilic moieties.

[0175] The oligomer may further comprise one or more lipophilic moietiesas will be understood by those skilled in the art. The lipophilic moietyis preferably a saturated or unsaturated, linear or branched alkylmoiety or a saturated or unsaturated, linear or branched fatty acidmoiety. When the lipophilic moiety is an alkyl moiety, it is preferablya linear, saturated or unsaturated alkyl moiety having 1 to 28 carbonatoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. Whenthe lipophilic moiety is a fatty acid moiety, it is preferably a naturalfatty acid moiety that is linear, saturated or unsaturated, having 2 to18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5or 6 carbon atoms.

[0176] The oligomer may further comprise one or more spacer moieties aswill be understood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from theinsulin drug, to separate a first hydrophilic or lipophilic moiety froma second hydrophilic or lipophilic moiety, or to separate a hydrophilicmoiety or lipophilic moiety from a linker moiety. Spacer moieties arepreferably selected from the group consisting of sugar, cholesterol andglycerine moieties.

[0177] The oligomer may further comprise one or more linker moietiesthat are used to couple the oligomer with the insulin drug as will beunderstood by those skilled in the art. Linker moieties are preferablyselected from the group consisting of alkyl and fatty acid moieties.

[0178] The oligomer may further comprise one or more terminatingmoieties at the one or more ends of the oligomer which are not coupledto the insulin drug. The terminating moiety is preferably an alkyl oralkoxy moiety, and is more preferably a lower alkyl or lower alkoxymoiety. Most preferably, the terminating moiety is methyl or methoxy.While the terminating moiety is preferably an alkyl or alkoxy moiety, itis to be understood that the terminating moiety may be various moietiesas will be understood by those skilled in the art including, but notlimited to, sugars, cholesterol, alcohols, and fatty acids.

[0179] The oligomer is preferably covalently coupled to the insulindrug. In some embodiments, the insulin drug is coupled to the oligomerutilizing a hydrolyzable bond (e.g., an ester or carbonate bond). Ahydrolyzable coupling may provide an insulin drug-oligomer conjugatethat acts as a prodrug. In certain instances, for example where theinsulin drug-oligomer conjugate is inactive (i.e., the conjugate lacksthe ability to affect the body through the insulin drug's primarymechanism of action), a hydrolyzable coupling may provide for atime-release or controlled-release effect, administering the insulindrug over a given time period as one or more oligomers are cleaved fromtheir respective insulin drug-oligomer conjugates to provide the activedrug. In other embodiments, the insulin drug is coupled to the oligomerutilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or etherbond). Use of a non-hydrolyzable bond may be preferable when it isdesirable to allow the insulin drug-oligomer conjugate to circulate inthe bloodstream for an extended period of time, preferably at least 2hours. When the oligomer is covalently coupled to the insulin drug, theoligomer further comprises one or more bonding moieties that are used tocovalently couple the oligomer with the insulin drug as will beunderstood by those skilled in the art. Bonding moieties are preferablyselected from the group consisting of covalent bond(s), ester moieties,carbonate moieties, carbamate moieties, amide moieties and secondaryamine moieties. More than one moiety on the oligomer may be covalentlycoupled to the insulin drug.

[0180] While the oligomer is preferably covalently coupled to theinsulin drug, it is to be understood that the oligomer may benon-covalently coupled to the insulin drug to form a non-covalentlyconjugated insulin drug-oligomer complex. As will be understood by thoseskilled in the art, non-covalent couplings include, but are not limitedto, hydrogen bonding, ionic bonding, Van der Waals bonding, andmicellular or liposomal encapsulation. According to embodiments of thepresent invention, oligomers may be suitably constructed, modifiedand/or appropriately functionalized to impart the ability fornon-covalent conjugation in a selected manner (e.g., to impart hydrogenbonding capability), as will be understood by those skilled in the art.According to other embodiments of present invention, oligomers may bederivatized with various compounds including, but not limited to, aminoacids, oligopeptides, peptides, bile acids, bile acid derivatives, fattyacids, fatty acid derivatives, salicylic acids, salicylic acidderivatives, aminosalicylic acids, and aminosalicylic acid derivatives.The resulting oligomers can non-covalently couple (complex) with drugmolecules, pharmaceutical products, and/or pharmaceutical excipients.The resulting complexes preferably have balanced lipophilic andhydrophilic properties. According to still other embodiments of thepresent invention, oligomers may be derivatized with amine and/or alkylamines. Under suitable acidic conditions, the resulting oligomers canform non-covalently conjugated complexes with drug molecules,pharmaceutical products and/or pharmaceutical excipients. The productsresulting from such complexation preferably have balanced lipophilic andhydrophilic properties.

[0181] More than one oligomer (i.e., a plurality of oligomers) may becoupled to the insulin drug. The oligomers in the plurality arepreferably the same. However, it is to be understood that the oligomersin the plurality may be different from one another, or, alternatively,some of the oligomers in the plurality may be the same and some may bedifferent. When a plurality of oligomers are coupled to the insulindrug, it may be preferable to couple one or more of the oligomers to theinsulin drug with hydrolyzable bonds and couple one or more of theoligomers to the insulin drug with non-hydrolyzable bonds.Alternatively, all of the bonds coupling the plurality of oligomers tothe insulin drug may be hydrolyzable, but have varying degrees ofhydrolyzability such that, for example, one or more of the oligomers israpidly removed from the insulin drug by hydrolysis in the body and oneor more of the oligomers is slowly removed from the insulin drug byhydrolysis in the body.

[0182] The oligomer may be coupled to the insulin drug at variousnucleophilic residues of the insulin drug including, but not limited to,nucleophilic hydroxyl functions and/or amino functions. When the insulindrug is a polypeptide, a nucleophilic hydroxyl function may be found,for example, at serine and/or tyrosine residues, and a nucleophilicamino function may be found, for example, at histidine and/or lysineresidues, and/or at the one or more N-termini of the polypeptide. Whenan oligomer is coupled to the one or more N-termini of the insulinpolypeptide, the coupling preferably forms a secondary amine. When theinsulin drug is human insulin, for example, the oligomer may be coupledto an amino functionality of the insulin, including the aminofunctionality of Gly^(A1), the amino functionality of Phe^(B1), and theamino functionality of Lys^(B29). When one oligomer is coupled to thehuman insulin, the oligomer is preferably coupled to the aminofunctionality of Lys^(B29). When two oligomers are coupled to the humaninsulin, the oligomers are preferably coupled to the amino functionalityof Phe^(B1) and the amino functionality of Lys^(B29); While more thanone oligomer may be coupled to the human insulin, a higher activity(improved glucose lowering ability) is observed for the mono-conjugatedhuman insulin.

[0183] Mixtures of insulin drug-oligomer conjugates where each conjugatein the mixture has the same number of polyethylene glycol subunits maybe synthesized by various methods. For example, a mixture of oligomersconsisting of carboxylic acid and polyethylene glycol where eacholigomer in the mixture has the same number of polyethylene glycolsubunits is synthesized by contacting a mixture of carboxylic acid witha mixture of polyethylene glycol where each polyethylene glycol moleculein the mixture has the same number of polyethylene glycol subunits underconditions sufficient to provide a mixture of oligomers where eacholigomer in the mixture has the same number of polyethylene glycolsubunits. The oligomers of the mixture where each oligomer in themixture has the same number of polyethylene glycol subunits are thenactivated so that they are capable of reacting with an insulin drug toprovide an insulin drug-oligomer conjugate. One embodiment of asynthesis route for providing a mixture of activated oligomers whereeach oligomer in the mixture has the same number of polyethylene glycolsubunits is illustrated in FIG. 3 and described in Examples 11-18hereinbelow. Another embodiment of a synthesis route for providing amixture of activated oligomers where each oligomer in the mixture hasthe same number of polyethylene glycol subunits is illustrated in FIG. 4and described in Examples 19-24 hereinbelow. Still another embodiment ofa synthesis route for providing a mixture of activated oligomers whereeach oligomer in the mixture has the same number of polyethylene glycolsubunits is illustrated in FIG. 5 and described in Examples 25-29hereinbelow. Yet another embodiment of a synthesis route for providing amixture of activated oligomers where each oligomer in the mixture hasthe same number of polyethylene glycol subunits is illustrated in FIG. 6and described in Examples 30-31 hereinbelow. Another embodiment of asynthesis route for providing a mixture of activated oligomers whereeach oligomer in the mixture has the same number of polyethylene glycolsubunits is illustrated in FIG. 7 and described in Examples 32-37hereinbelow. Still another embodiment of a synthesis route for providinga mixture of activated oligomers where each oligomer in the mixture hasthe same number of polyethylene glycol subunits is illustrated in FIG. 8and described in Example 38 hereinbelow. Yet another embodiment of asynthesis route for providing a mixture of activated oligomers whereeach oligomer in the mixture has the same number of polyethylene glycolsubunits is illustrated in FIG. 9 and described in Example 39hereinbelow. Another embodiment of a synthesis route for providing amixture of activated oligomers having a mixture of activated oligomerswhere each oligomer in the mixture has the same number of polyethyleneglycol subunits is illustrated in FIG. 10 and described in Example 40hereinbelow.

[0184] The mixture of activated oligomers where each oligomer in themixture has the same number of polyethylene glycol subunits is reactedwith a mixture of insulin drugs under conditions sufficient to provide amixture of insulin drug-oligomer conjugates. A preferred synthesis isdescribed in Example 41 hereinbelow. As will be understood by thoseskilled in the art, the reaction conditions (e.g., selected molarratios, solvent mixtures and/or pH) may be controlled such that themixture of insulin drug-oligomer conjugates resulting from the reactionof the mixture of activated oligomers where each oligomer in the mixturehas the same number of polyethylene glycol subunits and the mixture ofinsulin drugs is a mixture of conjugates where each conjugate in themixture has the same number of polyethylene glycol subunits. Forexample, conjugation at the amino functionality of lysine may besuppressed by maintaining the pH of the reaction solution below thepK_(a) of lysine. Alternatively, the mixture of insulin drug-oligomerconjugates may be separated and isolated utilizing, for example, HPLC asdescribed in Example 50 hereinbelow to provide a mixture of insulindrug-oligomer conjugates, for example mono-, di-, or tri-conjugates,where each conjugate in the mixture has the same number of polyethyleneglycol subunits. The degree of conjugation (e.g., whether the isolatedmolecule is a mono-, di-, or tri-conjugate) of a particular isolatedconjugate may be determined and/or verified utilizing various techniquesas will be understood by those skilled in the art including, but notlimited to, mass spectroscopy. The particular conjugate structure (e.g.,whether the oligomer is at Gly^(A1), Phe^(B1) or Lys^(B29) of a humaninsulin monoconjugate) may be determined and/or verified utilizingvarious techniques as will be understood by those skilled in the artincluding, but not limited to, sequence analysis, peptide mapping,selective enzymatic cleavage, and/or endopeptidase cleavage.

[0185] As will be understood by those skilled in the art, one or more ofthe reaction sites on the insulin drug may be blocked by, for example,reacting the insulin drug with a suitable blocking reagent such asN-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl)(N-FMOC). This process may be preferred, for example, when the insulindrug is a polypeptide and it is desired to form an unsaturated conjugate(i.e., a conjugate wherein not all nucleophilic residues are conjugated)having an oligomer at the one or more N-termini of the polypeptide.Following such blocking, the mixture of blocked insulin drugs may bereacted with the mixture of activated oligomers where each oligomer inthe mixture has the same number of polyethylene glycol subunits toprovide a mixture of insulin drug-oligomer conjugates having oligomer(s)coupled to one or more nucleophilic residues and having blockingmoieties coupled to other nucleophilic residues. After the conjugationreaction, the insulin drug-oligomer conjugates may be de-blocked as willbe understood by those skilled in the art. If necessary, the mixture ofinsulin drug-oligomer conjugates may then be separated as describedabove to provide a mixture of insulin drug-oligomer conjugates whereeach conjugate in the mixture has the same number of polyethylene glycolsubunits. Alternatively, the mixture of insulin drug-oligomer conjugatesmay be separated prior to de-blocking.

[0186] Mixtures of insulin drug-oligomer conjugates where each conjugatein the mixture has the same number of polyethylene glycol subunitsaccording to these embodiments of the present invention preferably haveimproved properties when compared with those of polydispersed mixtures.For example, a mixture of insulin drug-oligomer conjugates where eachconjugate in the mixture has the same number of polyethylene glycolsubunits preferably has an in vivo activity that is greater than the invivo activity of a polydispersed mixture of insulin drug-oligomerconjugates having the same number average molecular weight as themixture of insulin drug-oligomer conjugates where each conjugate in themixture has the same number of polyethylene glycol subunits. As will beunderstood by those skilled in the art, the number average molecularweight of the mixture of insulin drug-oligomer conjugates where eachconjugate in the mixture has the same number of polyethylene glycolsubunits and the number average weight of the polydispersed mixture maybe measured by various methods including, but not limited to, sizeexclusion chromatography such as gel permeation chromatography asdescribed, for example, in H. R. Allcock & F. W. Lampe, CONTEMPORARYPOLYMER CHEMISTRY 394-402 (2d. ed., 1991).

[0187] As another example, a mixture of insulin drug-oligomer conjugateswhere each conjugate in the mixture has the same number of polyethyleneglycol subunits preferably has an in vitro activity that is greater thanthe in vitro activity of a polydispersed mixture of insulindrug-oligomer conjugates having the same number average molecular weightas the mixture of insulin drug-oligomer conjugates where each conjugatein the mixture has the same number of polyethylene glycol subunits. Aswill be understood by those skilled in the art, the number averagemolecular weight of the mixture of insulin drug-oligomer conjugateswhere each conjugate in the mixture has the same number of polyethyleneglycol subunits and the number average weight of the polydispersedmixture may be measured by various methods including, but not limitedto, size exclusion chromatography.

[0188] The in vitro activity of a particular mixture may be measured byvarious methods, as will be understood by those skilled in the art.Preferably, the in vitro activity is measured using a Cytosensor®Microphysiometer commercially available from Molecular DevicesCorporation of Sunnyvale, Calif. The microphysiometer monitors smallchanges in the rates of extracellular acidification in response to adrug being added to cultured cells in a transwell. This response isproportional to the activity of the molecule under study. Preferably,the in vitro activity of the mixture of insulin drug-oligomer conjugateswhere each conjugate in the mixture has the same number of polyethyleneglycol subunits is at least about 5 percent greater than the in vitroactivity of the polydispersed mixture. More preferably, the in vitroactivity of the mixture of insulin drug-oligomer conjugates where eachconjugate in the mixture has the same number of polyethylene glycolsubunits is at least about 10 percent greater than the in vitro activityof the polydispersed mixture.

[0189] As still another example, a mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same number ofpolyethylene glycol subunits preferably has an increased resistance todegradation by chymotrypsin when compared to the resistance todegradation by chymotrypsin of a polydispersed mixture of insulindrug-oligomer conjugates having the same number average molecular weightas the mixture of insulin drug-oligomer conjugates where each conjugatein the mixture has the same number of polyethylene glycol subunits.Resistance to chymotrypsin corresponds to the percent remaining when themolecule to be tested is digested in chymotrypsin using a proceduresimilar to the one outlined in Example 52 below. As will be understoodby those skilled in the art, the number average molecular weight of themixture of insulin drug-oligomer conjugates where each conjugate in themixture has the same number of polyethylene glycol subunits and thenumber average weight of the polydispersed mixture may be measured byvarious methods including, but not limited to, size exclusionchromatography. Preferably, the resistance to degradation bychymotrypsin of the mixture of insulin drug-oligomer conjugates whereeach conjugate in the mixture has the same number of polyethylene glycolsubunits is at least about 10 percent greater than the resistance todegradation by chymotrypsin of the polydispersed mixture. Morepreferably, the resistance to degradation by chymotrypsin of the mixtureof insulin drug-oligomer conjugates where each conjugate in the mixturehas the same number of polyethylene glycol subunits is at least about 20percent greater than the resistance to degradation by chymotrypsin ofthe polydispersed mixture.

[0190] As yet another example, a mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same number ofpolyethylene glycol subunits preferably has an inter-subject variabilitythat is less than the inter-subject variability of a polydispersedmixture of insulin drug-oligomer conjugates having the same numberaverage molecular weight as the mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same number ofpolyethylene glycol subunits. As will be understood by those skilled inthe art, the number average molecular weight of the mixture of insulindrug-oligomer conjugates where each conjugate in the mixture has thesame number of polyethylene glycol subunits and the number averageweight of the polydispersed mixture may be measured by various methodsincluding, but not limited to, size exclusion chromatography. Theinter-subject variability may be measured by various methods as will beunderstood by those skilled in the art. The inter-subject variability ispreferably calculated as follows. The area under a dose response curve(AUC) (i.e., the area between the dose-response curve and a baselinevalue) is determined for each subject. The average AUC for all subjectsis determined by summing the AUCs of each subject and dividing the sumby the number of subjects. The absolute value of the difference betweenthe subject's AUC and the average AUC is then determined for eachsubject. The absolute values of the differences obtained are then summedto give a value that represents the inter-subject variability. Lowervalues represent lower inter-subject variabilities and higher valuesrepresent higher inter-subject variabilities. Preferably, theinter-subject variability of the mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same number ofpolyethylene glycol subunits is at least about 10 percent less than theinter-subject variability of the polydispersed mixture. More preferably,the inter-subject variability of the mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same number ofpolyethylene glycol subunits is at least about 25 percent less than theinter-subject variability of the polydispersed mixture.

[0191] Mixtures of insulin drug-oligomer conjugates where each conjugatein the mixture has the same number of polyethylene glycol subunitsaccording to embodiments of the present invention preferably have two ormore of the above-described properties. More preferably, mixtures ofinsulin drug-oligomer conjugates where each conjugate in the mixture hasthe same number of polyethylene glycol subunits according to embodimentsof the present invention have three or more of the above-describedproperties. Most preferably, mixtures of insulin drug-oligomerconjugates where each conjugate in the mixture has the same number ofpolyethylene glycol subunits according to embodiments of the presentinvention have all four of the above-described properties.

[0192] According to still other embodiments of the present invention, amixture of conjugates is provided in which each conjugate has the samemolecular weight and has the structure of Formula A:

Insulin DrugB-L_(j)-G_(k)-R-G′_(m)-R′-G″_(n)-T]_(p)   (A)

[0193] wherein:

[0194] B is a bonding moiety;

[0195] L is a linker moiety;

[0196] G, G′ and G″ are individually selected spacer moieties;

[0197] R is a lipophilic moiety and R′ is a polyalkylene glycol moiety,or R′ is the lipophilic moiety and R is the polyalkylene glycol moiety;

[0198] T is a terminating moiety;

[0199] j, k, m and n are individually 0 or 1; and

[0200] p is an integer between 1 and the number of nucleophilic residueson the insulin drug.

[0201] The insulin drug is preferably insulin. More preferably, theinsulin drug is human insulin. However, it is to be understood that theinsulin drug may be selected from various insulin drugs known to thoseskilled in the art including, for example, proinsulin, insulin analogs,insulin fragments, and insulin fragment analogs. Insulin analogsinclude, but are not limited to, Asp^(B28) human insulin, Lys^(B28)human insulin, Leu^(B28) human insulin, Val^(B28) human insulin,Ala^(B28) human insulin, Asp^(B28)Pro^(B29) human insulin,Lys^(B28)Pro^(B29) human insulin, Leu^(B28)Pro^(B29) human insulin,Val^(B28)Pro^(B29) human insulin, Ala^(B28)Pro^(B29) human insulin, aswell as analogs provided using the substitution guidelines describedabove. Insulin fragments include, but are not limited to, B22-B30 humaninsulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 humaninsulin, B27-B30 human insulin, B29-B30 human insulin, the A chain ofhuman insulin, and the B chain of human insulin. Insulin fragmentanalogs may be provided by substituting one or more amino acids asdescribed above in an insulin fragment.

[0202] According to these embodiments of the present invention, thepolyalkylene glycol moiety of the oligomer preferably has at least 2, 3or 4 polyalkylene glycol subunits. More preferably, the polyalkyleneglycol moiety has at least 5 or 6 polyalkylene glycol subunits and, mostpreferably, the polyethylene glycol moiety has at least 7 polyalkyleneglycol subunits. The polyalkylene glycol moiety is preferably a lowerpolyalkylene glycol moiety such as a polyethylene glycol moiety, apolypropylene glycol moiety, or a polybutylene glycol moiety. Morepreferably, the polyalkylene glycol moiety is a polyethylene glycolmoiety or a polypropylene glycol moiety. Most preferably, thepolyalkylene glycol moiety is a polyethylene glycol moiety. When thepolyalkylene glycol moiety is a polypropylene glycol moiety, the moietypreferably has a uniform (i.e., not random) structure. An exemplarypolypropylene glycol moiety having a uniform structure is as follows:

[0203] This uniform polypropylene glycol structure may be described ashaving only one methyl substituted carbon atom adjacent each oxygen atomin the polypropylene glycol chain. Such uniform polypropylene glycolmoieties may exhibit both lipophilic and hydrophilic characteristics andthus be useful in providing amphiphilic insulin drug-oligomer conjugateswithout the use of lipophilic polymer moieties. Furthermore, couplingthe secondary alcohol moiety of the polypropylene glycol moiety with andrug may provide the insulin drug (e.g., human insulin) with improvedresistance to degradation caused by enzymes such as trypsin andchymotrypsin found, for example, in the gut.

[0204] Uniform polypropylene glycol according to embodiments of thepresent invention is preferably synthesized as illustrated in FIGS. 11through 13, which will now be described. As illustrated in FIG. 11,1,2-propanediol 53 is reacted with a primary alcohol blocking reagent toprovide a secondary alcohol extension monomer 54. The primary alcoholblocking reagent may be various primary alcohol blocking reagents aswill be understood by those skilled in the art including, but notlimited to, silylchloride compounds such as t-butyldiphenylsilylchlorideand t-butyldimethylsilylchloride, and esterification reagents such asAc₂O. Preferably, the primary alcohol blocking reagent is a primaryalcohol blocking reagent that is substantially non-reactive withsecondary alcohols, such as t-butyldiphenylsilylchloride ort-butyldimethylsilylchloride. The secondary alcohol extension monomer(54) may be reacted with methanesulfonyl chloride (MeSO₂Cl) to provide aprimary extension alcohol monomer mesylate 55.

[0205] Alternatively, the secondary alcohol extension monomer 54 may bereacted with a secondary alcohol blocking reagent to provide compound56. The secondary alcohol blocking reagent may be various secondaryalcohol blocking reagents as will be understood by those skilled in theart including, but not limited to, benzyl chloride. The compound 56 maybe reacted with a B₁ de-blocking reagent to remove the blocking moietyB₁ and provide a primary alcohol extension monomer 57. The B₁de-blocking reagent may be selected from various de-blocking reagents aswill be understood by one skilled in the art. When the primary alcoholhas been blocked by forming an ester, the B₁ de-blocking reagent is ade-esterification reagent, such as a base (e.g., potassium carbonate).When the primary alcohol has been blocked using a silylchloride, the B₁de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).The primary alcohol extension monomer 57 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension monomermesylate 58.

[0206] The primary alcohol extension monomer 54 and the secondaryalcohol extension monomer 57 may be capped as follows. The secondaryalcohol extension monomer 54 may be reacted with a capping reagent toprovide a compound 59. The capping reagent may be various cappingreagents as will be understood by those skilled in the art including,but not limited to, alkyl halides such as methyl chloride. The compound59 may be reacted with a B₁ de-blocking agent as described above toprovide a primary alcohol capping monomer 60. The primary alcoholcapping monomer 60 may be reacted with methane sulfonyl chloride toprovide the secondary alcohol capping monomer mesylate 61. The primaryalcohol extension monomer 57 may be reacted with a capping reagent toprovide a compound 62. The capping reagent may be various cappingreagents as described above. The compound 62 may be reacted with a B₂de-blocking reagent to remove the blocking moiety B₂ and provide asecondary alcohol capping monomer 63. The B₂ de-blocking reagent may bevarious de-blocking agents as will be understood by those skilled in theart including, but not limited to, H₂ in the presence of apalladium/activated carbon catalyst. The secondary alcohol cappingmonomer may be reacted with methanesulfonyl chloride to provide aprimary alcohol capping monomer mesylate 64. While the embodimentsillustrated in FIG. 11 show the synthesis of capping monomers, it is tobe understood that similar reactions may be performed to provide cappingpolymers.

[0207] In general, chain extensions may be effected by reacting aprimary alcohol extension mono- or poly-mer such as the primary alcoholextension monomer 57 with a primary alcohol extension mono- or poly-mermesylate such as the primary alcohol extension monomer mesylate 55 toprovide various uniform polypropylene chains or by reacting a secondaryalcohol extension mono- or poly-mer such as the secondary alcoholextension monomer 54 with a secondary alcohol extension mono-or poly-mermesylate such as the secondary alcohol extension monomer mesylate 58.

[0208] For example, in FIG. 13, the primary alcohol extension monomermesylate 55 is reacted with the primary alcohol extension monomer 57 toprovide a dimer compound 65. Alternatively, the secondary alcoholextension monomer mesylate 58 may be reacted with the secondary alcoholextension monomer 54 to provide the dimer compound 65. The B₁ blockingmoiety on the dimer compound 65 may be removed using a B₁ de-blockingreagent as described above to provide a primary alcohol extension dimer66. The primary alcohol extension dimer 66 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension dimermesylate 67. Alternatively, the B₂ blocking moiety on the dimer compound65 may be removed using the B₂ de-blocking reagent as described above toprovide a secondary alcohol extension dimer 69. The secondary alcoholextension dimer 69 may be reacted with methane sulfonyl chloride toprovide a primary alcohol extension dimer mesylate 70.

[0209] As will be understood by those skilled in the art, the chainextension process may be repeated to achieve various other chainlengths. For example, as illustrated in FIG. 13, the primary alcoholextension dimer 66 may be reacted with the primary alcohol extensiondimer mesylate 70 to provide a tetramer compound 72. As furtherillustrated in FIG. 13, a generic chain extension reaction schemeinvolves reacting the primary alcohol extension mono- or poly-mer 73with the primary alcohol extension mono- or poly-mer mesylate 74 toprovide the uniform polypropylene polymer 75. The values of m and n mayeach range from 0 to 1000 or more. Preferably, m and n are each from 0to 50. While the embodiments illustrated in FIG. 13 show primary alcoholextension mono- and/or poly-mers being reacted with primary alcoholextension mono- and/or poly-mer mesylates, it is to be understood thatsimilar reactions may be carried out using secondary alcohol extensionmono- and/or poly-mers and secondary alcohol extension mono- and/orpoly-mer mesylates.

[0210] An end of a primary alcohol extension mono- or poly-mer or an endof a primary alcohol extension mono- or poly-mer mesylate may be reactedwith a primary alcohol capping mono- or poly-mer mesylate or a primaryalcohol capping mono- or poly-mer, respectively, to provide a cappeduniform polypropylene chain. For example, as illustrated in FIG. 12, theprimary alcohol extension dimer mesylate 70 is reacted with the primaryalcohol capping monomer 60 to provide the capped/blocked primary alcoholextension trimer 71. As will be understood by those skilled in the art,the B₁ blocking moiety may be removed and the resulting capped primaryalcohol extension trimer may be reacted with a primary alcohol extensionmono- or poly-mer mesylate to extend the chain of the capped trimer 71.

[0211] An end of a secondary alcohol extension mono-or poly-mer or anend of a secondary alcohol extension mono-or poly-mer mesylate may bereacted with a secondary alcohol capping mono-or poly-mer mesylate or asecondary alcohol capping mono- or poly-mer, respectively, to provide acapped uniform polypropylene chain. For example, as illustrated in FIG.12, the secondary alcohol extension dimer mesylate 67 is reacted withthe secondary alcohol capping monomer 63 to provide the capped/blockedprimary alcohol extension trimer 68. The B₂ blocking moiety may beremoved as described above and the resulting capped secondary alcoholextension trimer may be reacted with a secondary alcohol extension mermesylate to extend the chain of the capped trimer 68. While thesyntheses illustrated in FIG. 12 show the reaction of a dimer with acapping monomer to provide a trimer, it is to be understood that thecapping process may be performed at any point in the synthesis of auniform polypropylene glycol moiety, or, alternatively, uniformpolypropylene glycol moieties may be provided that are not capped. Whilethe embodiments illustrated in FIG. 12 show the capping of apolybutylene oligomer by synthesis with a capping monomer, it is to beunderstood that polybutylene oligomers of the present invention may becapped directly (i.e., without the addition of a capping monomer) usinga capping reagent as described above in FIG. 11.

[0212] Uniform polypropylene glycol moieties according to embodiments ofthe present invention may be coupled to an insulin drug, a lipophilicmoiety such as a carboxylic acid, and/or various other moieties byvarious methods as will be understood by those skilled in the artincluding, but not limited to, those described herein with respect topolyethylene glycol moieties.

[0213] According to these embodiments of the present invention, thelipophilic moiety is a lipophilic moiety as will be understood by thoseskilled in the art. The lipophilic moiety is preferably a saturated orunsaturated, linear or branched alkyl moiety or a saturated orunsaturated, linear or branched fatty acid moiety. When the lipophilicmoiety is an alkyl moiety, it is preferably a linear, saturated orunsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably,the alkyl moiety has 2 to 12 carbon atoms. When the lipophilic moiety isa fatty acid moiety, it is preferably a natural fatty acid moiety thatis linear, saturated or unsaturated, having 2 to 18 carbon atoms. Morepreferably, the fatty acid moiety has 3 to 14 carbon atoms. Mostpreferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.

[0214] According to these embodiments of the present invention, thespacer moieties, G, G′ and G″, are spacer moieties as will be understoodby those skilled in the art. Spacer moieties are preferably selectedfrom the group consisting of sugar, cholesterol and glycerine moieties.Preferably, oligomers of these embodiments do not include spacermoieties (i.e., k, m and n are preferably 0).

[0215] According to these embodiments of the present invention, thelinker moiety, L, may be used to couple the oligomer with the drug aswill be understood by those skilled in the art. Linker moieties arepreferably selected from the group consisting of alkyl and fatty acidmoieties.

[0216] According to these embodiments of the present invention, theterminating moiety is preferably an alkyl or alkoxy moiety, and is morepreferably a lower alkyl or lower alkoxy moiety. Most preferably, theterminating moiety is methyl or methoxy. While the terminating moiety ispreferably an alkyl or alkoxy moiety, it is to be understood that theterminating moiety may be various moieties as will be understood bythose skilled in the art including, but not limited to, sugars,cholesterol, alcohols, and fatty acids.

[0217] According to these embodiments of the present invention, theoligomer, which is represented by the bracketed portion of the structureof Formula A, is covalently coupled to the insulin drug. In someembodiments, the insulin drug is coupled to the oligomer utilizing ahydrolyzable bond (e.g., an ester or carbonate bond). A hydrolyzablecoupling may provide an insulin drug-oligomer conjugate that acts as aprodrug. In certain instances, for example where the insulindrug-oligomer conjugate is inactive (i.e., the conjugate lacks theability to affect the body through the insulin drug's primary mechanismof action), a hydrolyzable coupling may provide for a time-release orcontrolled-release effect, administering the insulin drug over a giventime period as one or more oligomers are cleaved from their respectiveinsulin drug-oligomer conjugates to provide the active drug. In otherembodiments, the insulin drug is coupled to the oligomer utilizing anon-hydrolyzable bond (e.g., a carbamate, amide, or ether bond). Use ofa non-hydrolyzable bond may be preferable when it is desirable to allowthe insulin drug-oligomer conjugate to circulate in the bloodstream foran extended period of time, preferably at least 2 hours. The bondingmoiety, B, may be various bonding moieties as will be understood bythose skilled in the art. Bonding moieties are preferably selected fromthe group consisting of covalent bond(s), ester moieties, carbonatemoieties, carbamate moieties, amide moieties and secondary aminemoieties.

[0218] The variable p is an integer from 1 to the number of nucleophilicresidues on the insulin drug. When p is greater than 1, more than oneoligomer (i.e., a plurality of oligomers) is coupled to the drug.According to these embodiments of the present invention, the oligomersin the plurality are the same. When a plurality of oligomers are coupledto the insulin drug, it may be preferable to couple one or more of theoligomers to the insulin drug with hydrolyzable bonds and couple one ormore of the oligomers to the insulin drug with non-hydrolyzable bonds.Alternatively, all of the bonds coupling the plurality of oligomers tothe insulin drug may be hydrolyzable, but have varying degrees ofhydrolyzability such that, for example, one or more of the oligomers israpidly removed from the insulin drug by hydrolysis in the body and oneor more of the oligomers is slowly removed from the insulin drug byhydrolysis in the body.

[0219] The oligomer may be coupled to the insulin drug at variousnucleophilic residues of the insulin drug including, but not limited to,nucleophilic hydroxyl functions and/or amino functions. When the insulindrug is a polypeptide, a nucleophilic hydroxyl function may be found,for example, at serine and/or tyrosine residues, and a nucleophilicamino function may be found, for example, at histidine and/or lysineresidues, and/or at the one or more N-termini of the polypeptide. Whenan oligomer is coupled to the one or more N-termini of the insulinpolypeptide, the coupling preferably forms a secondary amine. When theinsulin drug is human insulin, for example, the oligomer may be coupledto an amino functionality of the insulin, including the aminofunctionality of Gly^(A1), the amino functionality of Phe^(B1), and theamino functionality of Lys^(B29). When one oligomer is coupled to thehuman insulin, the oligomer is preferably coupled to the aminofunctionality of Lys^(B29). When two oligomers are coupled to the humaninsulin, the oligomers are preferably coupled to the amino functionalityof Phe^(B1) and the amino functionality of Lys^(B29). While more thanone oligomer may be coupled to the human insulin, a higher activity(improved glucose lowering ability) is observed for the mono-conjugatedhuman insulin.

[0220] Mixtures of insulin drug-oligomer conjugates where each conjugatein the mixture has the same molecular weight and has the structure ofFormula A may be synthesized by various methods. For example, a mixtureof oligomers consisting of carboxylic acid and polyethylene glycol issynthesized by contacting a mixture of carboxylic acid with a mixture ofpolyethylene glycol under conditions sufficient to provide a mixture ofoligomers. The oligomers of the mixture are then activated so that theyare capable of reacting with an insulin drug to provide an insulindrug-oligomer conjugate. One embodiment of a synthesis route forproviding a mixture of activated oligomers where each oligomer has thesame molecular weight and has a structure of the oligomer of Formula Ais illustrated in FIG. 3 and described in Examples I1-18 hereinbelow.Another embodiment of a synthesis route for providing a mixture ofactivated oligomers where each oligomer has the same molecular weightand has a structure of the oligomer of Formula A is illustrated in FIG.4 and described in Examples 19-24 hereinbelow. Still another embodimentof a synthesis route for providing a mixture of activated oligomerswhere each oligomer has the same molecular weight and has a structure ofthe oligomer of Formula A is illustrated in FIG. 5 and described inExamples 25-29 hereinbelow. Yet another embodiment of a synthesis routefor providing a mixture of activated oligomers where each oligomer hasthe same molecular weight and has a structure of the oligomer of FormulaA is illustrated in FIG. 6 and described in Examples 30-31 hereinbelow.Another embodiment of a synthesis route for providing a mixture ofactivated oligomers where each oligomer has the same molecular weightand has a structure of the oligomer of Formula A is illustrated in FIG.7 and described in Examples 32-37 hereinbelow. Still another embodimentof a synthesis route for providing a mixture of activated oligomerswhere each oligomer has the same molecular weight and has a structure ofthe oligomer of Formula A is illustrated in FIG. 8 and described inExample 38 hereinbelow. Yet another embodiment of a synthesis route forproviding a mixture of activated oligomers where each oligomer has thesame molecular weight and has a structure of the oligomer of Formula Ais illustrated in FIG. 9 and described in Example 39 hereinbelow.Another embodiment of a synthesis route for providing a mixture ofactivated oligomers where each oligomer has the same molecular weightand has a structure of the oligomer of Formula A is illustrated in FIG.10 and described in Example 40 hereinbelow.

[0221] The mixture of activated oligomers where each oligomer has thesame molecular weight and has a structure of the oligomer of Formula Ais reacted with a mixture of insulin drugs where each drug in themixture has the same molecular weight under conditions sufficient toprovide a mixture of insulin drug-oligomer conjugates. A preferredsynthesis is described in Example 41 hereinbelow. As will be understoodby those skilled in the art, the reaction conditions (e.g., selectedmolar ratios, solvent mixtures and/or pH) may be controlled such thatthe mixture of insulin drug-oligomer conjugates resulting from thereaction of the mixture of activated oligomers where each oligomer hasthe same molecular weight and has a structure of the oligomer of FormulaA and the mixture of insulin drugs is a mixture of conjugates where eachconjugate has the same molecular weight and has the structure Formula A.For example, conjugation at the amino functionality of lysine may besuppressed by maintaining the pH of the reaction solution below thepK_(a) of lysine. Alternatively, the mixture of insulin drug-oligomerconjugates may be separated and isolated utilizing, for example, HPLC asdescribed below in Example 50 to provide a mixture of insulindrug-oligomer conjugates, for example mono-, di-, or tri-conjugates,where each conjugate in the mixture has the same number molecular weightand has the structure of Formula A. The degree of conjugation (e.g.,whether the isolated molecule is a mono-, di-, or tri-conjugate) of aparticular isolated conjugate may be determined and/or verifiedutilizing various techniques as will be understood by those skilled inthe art including, but not limited to, mass spectroscopy. The particularconjugate structure (e.g., whether the oligomer is at Gly^(A1), Phe^(B1)or Lys^(B29) of a human insulin monoconjugate) may be determined and/orverified utilizing various techniques as will be understood by thoseskilled in the art including, but not limited to, sequence analysis,peptide mapping, selective enzymatic cleavage, and/or endopeptidasecleavage.

[0222] As will be understood by those skilled in the art, one or more ofthe reaction sites on the insulin drug may be blocked by, for example,reacting the insulin drug with a suitable blocking reagent such asN-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl)(N-FMOC). This process may be preferred, for example, when the insulindrug is a polypeptide and it is desired to form an unsaturated conjugate(i.e., a conjugate wherein not all nucleophilic residues are conjugated)having an oligomer at the one or more N-termini of the polypeptide.Following such blocking, the mixture of blocked insulin drugs may bereacted with the mixture of activated oligomers where each oligomer inthe mixture has the same molecular weight and has a structure of theoligomer of Formula A to provide a mixture of insulin drug-oligomerconjugates having oligomer(s) coupled to one or more nucleophilicresidues and having blocking moieties coupled to other nucleophilicresidues. After the conjugation reaction, the insulin drug-oligomerconjugates may be de-blocked as will be understood by those skilled inthe art. If necessary, the mixture of insulin drug-oligomer conjugatesmay then be separated as described above to provide a mixture of insulindrug-oligomer conjugates where each conjugate in the mixture has thesame number molecular weight and has the structure of Formula A.Alternatively, the mixture of insulin drug-oligomer conjugates may beseparated prior to de-blocking.

[0223] Mixtures of insulin drug-oligomer conjugates where each conjugatein the mixture has the same molecular weight and has the structure ofFormula A according to these embodiments of the present inventionpreferably have improved properties when compared with those ofpolydispersed mixtures. For example, a mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same molecularweight and has the structure of Formula A preferably has an in vivoactivity that is greater than the in vivo activity of a polydispersedmixture of insulin drug-oligomer conjugates having the same numberaverage molecular weight as the mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same molecularweight and has the structure of Formula A. As will be understood bythose skilled in the art, the number average molecular weight of themixture of insulin drug-oligomer conjugates where each conjugate in themixture has the same molecular weight and has the structure of Formula Aand the number average weight of the polydispersed mixture may bemeasured by various methods including, but not limited to, sizeexclusion chromatography such as gel permeation chromatography asdescribed, for example, in H. R. Allcock & F. W. Lampe, CONTEMPORARYPOLYMER CHEMISTRY 394-402 (2d. ed., 1991).

[0224] As another example, a mixture of insulin drug-oligomer conjugateswhere each conjugate in the mixture has the same molecular weight andhas the structure of Formula A preferably has an in vitro activity thatis greater than the in vitro activity of a polydispersed mixture ofinsulin drug-oligomer conjugates having the same number averagemolecular weight as the mixture of insulin drug-oligomer conjugateswhere each conjugate in the mixture has the same molecular weight andhas the structure of Formula A. As will be understood by those skilledin the art, the number average molecular weight of the mixture ofinsulin drug-oligomer conjugates where each conjugate in the mixture hasthe same molecular weight and has the structure of Formula A and thenumber average weight of the polydispersed mixture may be measured byvarious methods including, but not limited to, size exclusionchromatography.

[0225] The in vitro activity of a particular mixture may be measured byvarious methods, as will be understood by those skilled in the art.Preferably, the in vitro activity is measured using a Cytosensor®Microphysiometer commercially available from Molecular DevicesCorporation of Sunnyvale, Calif. The microphysiometer monitors smallchanges in the rates of extracellular acidification in response to adrug being added to cultured cells in a transwell. This response isproportional to the activity of the molecule under study. Preferably,the in vitro activity of the mixture of insulin drug-oligomer conjugateswhere each conjugate in the mixture has the same molecular weight andhas the structure of Formula A is at least about 5 percent greater thanthe in vitro activity of the polydispersed mixture. More preferably, thein vitro activity of the mixture of insulin drug-oligomer conjugateswhere each conjugate in the mixture has the same molecular weight andhas the structure of Formula A is at least about 10 percent greater thanthe in vitro activity of the polydispersed mixture.

[0226] As still another example, a mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same molecularweight and has the structure of Formula A preferably has an increasedresistance to degradation by chymotrypsin when compared to theresistance to degradation by chymotrypsin of a polydispersed mixture ofinsulin drug-oligomer conjugates having the same~number averagemolecular weight as the mixture of insulin drug-oligomer conjugateswhere each conjugate in the mixture has the same molecular weight andhas the structure of Formula A. Resistance to chymotrypsin correspondsto the percent remaining when the molecule to be tested is digested inchymotrypsin using a procedure similar to the one outlined in Example 52below. As will be understood by those skilled in the art, the numberaverage molecular weight of the mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same molecularweight and has the structure of Formula A and the number average weightof the polydispersed mixture may be measured by various methodsincluding, but not limited to, size exclusion chromatography.Preferably, the resistance to degradation by chymotrypsin of the Amixture of insulin drug-oligomer conjugates where each conjugate in themixture has the same molecular weight and has the structure of Formula Ais at least about 10 percent greater than the resistance to degradationby chymotrypsin of the polydispersed mixture. More preferably, theresistance to degradation by chymotrypsin of the A mixture of insulindrug-oligomer conjugates where each conjugate in the mixture has thesame molecular weight and has the structure of Formula A is at leastabout 20 percent greater than the resistance to degradation bychymotrypsin of the polydispersed mixture.

[0227] As yet another example, a mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same molecularweight and has the structure of Formula A preferably has aninter-subject variability that is less than the inter-subjectvariability of a polydispersed mixture of insulin drug-oligomerconjugates having the same number average molecular weight as themixture of insulin drug-oligomer conjugates where each conjugate in themixture has the same molecular weight and has the structure of FormulaA. As will be understood by those skilled in the art, the number averagemolecular weight of the mixture of insulin drug-oligomer conjugateswhere each conjugate in the mixture has the same molecular weight andhas the structure of Formula A and the number average weight of thepolydispersed mixture may be measured by various methods including, butnot limited to, size exclusion chromatography. The inter-subjectvariability may be measured by various methods as will be understood bythose skilled in the art. The inter-subject variability is preferablycalculated as follows. The area under a dose response curve (AUC) (i.e.,the area between the dose-response curve and a baseline value) isdetermined for each subject. The average AUC for all subjects isdetermined by summing the AUCs of each subject and dividing the sum bythe number of subjects. The absolute value of the difference between thesubject's AUC and the average AUC is then determined for each subject.The absolute values of the differences obtained are then summed to givea value that represents the inter-subject variability. Lower valuesrepresent lower inter-subject variabilities and higher values representhigher inter-subject variabilities. Preferably, the inter-subjectvariability of the mixture of insulin drug-oligomer conjugates whereeach conjugate in the mixture has the same molecular weight and has thestructure of Formula A is at least about 10 percent less than theinter-subject variability of the polydispersed mixture. More preferably,the inter-subject variability of the mixture of insulin drug-oligomerconjugates where each conjugate in the mixture has the same molecularweight and has the structure of Formula A is at least about 25 percentless than the inter-subject variability of the polydispersed mixture.

[0228] Mixtures of insulin drug-oligomer conjugates where each conjugatein the mixture has the same molecular weight and has the structure ofFormula A according to embodiments of the present invention preferablyhave two or more of the above-described properties. More preferably,mixtures of insulin drug-oligomer conjugates where each conjugate in themixture has the same molecular weight and has the structure of Formula Aaccording to embodiments of the present invention have three or more ofthe above-described properties. Most preferably, mixtures of insulindrug-oligomer conjugates where each conjugate in the mixture has thesame molecular weight and has the structure of Formula A according toembodiments of the present invention have all four of theabove-described properties.

[0229] Pharmaceutical compositions comprising a conjugate mixtureaccording to embodiments of the present invention are also provided. Themixtures of insulin drug-oligomer conjugates described above may beformulated for administration in a pharmaceutical carrier in accordancewith known techniques. See, e.g., Remington, The Science And Practice ofPharmacy (9^(th) Ed. 1995). In the manufacture of a pharmaceuticalcomposition according to embodiments of the present invention, themixture of insulin drug-oligomer conjugates is typically admixed with,inter alia, a pharmaceutically acceptable carrier. The carrier must, ofcourse, be acceptable in the sense of being compatible with any otheringredients in the pharmaceutical composition and should not bedeleterious to the subject. The carrier may be a solid or a liquid, orboth, and is preferably formulated with the mixture of insulindrug-oligomer conjugates as a unit-dose formulation, for example, atablet, which may contain from about 0.01 or 0.5% to about 95% or 99% byweight of the mixture of insulin drug-oligomer conjugates. Thepharmaceutical compositions may be prepared by any of the well knowntechniques of pharmacy including, but not limited to, admixing thecomponents, optionally including one or more accessory ingredients.

[0230] The pharmaceutical compositions according to embodiments of thepresent invention include those suitable for oral, rectal, topical,inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal,parenteral (e.g., subcutaneous, intramuscular, intradermal,intraarticular, intrapleural, intraperitoneal, inracerebral,intraarterial, or intravenous), topical (i.e., both skin and mucosalsurfaces, including airway surfaces) and transdermal administration,although the most suitable route in any given case will depend on thenature and severity of the condition being treated and on the nature ofthe particular mixture of insulin drug-oligomer conjugates which isbeing used.

[0231] Pharmaceutical compositions suitable for oral administration maybe presented in discrete units, such as capsules, cachets, lozenges, ortables, each containing a predetermined amount of the mixture of insulindrug-oligomer conjugates; as a powder or granules; as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil emulsion. Such formulations may be prepared by any suitablemethod of pharmacy which includes the step of bringing into associationthe mixture of insulin drug-oligomer conjugates and a suitable carrier(which may contain one or more accessory ingredients as noted above). Ingeneral, the pharmaceutical composition according to embodiments of thepresent invention are prepared by uniformly and intimately admixing themixture of insulin drug-oligomer conjugates with a liquid or finelydivided solid carrier, or both, and then, if necessary, shaping theresulting mixture. For example, a tablet may be prepared by compressingor molding a powder or granules containing the mixture of insulindrug-oligomer conjugates, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing, in asuitable machine, the mixture in a free-flowing form, such as a powderor granules optionally mixed with a binder, lubricant, inert diluent,and/or surface active/dispersing agent(s). Molded tablets may be made bymolding, in a suitable machine, the powdered compound moistened with aninert liquid binder.

[0232] Pharmaceutical compositions suitable for buccal (sub-lingual)administration include lozenges comprising the mixture of insulindrug-oligomer conjugates in a flavoured base, usually sucrose and acaciaor tragacanth; and pastilles comprising-the mixture of insulindrug-oligomer conjugates in an inert base such as gelatin and glycerinor sucrose and acacia.

[0233] Pharmaceutical compositions according to embodiments of thepresent invention suitable for parenteral administration comprisesterile aqueous and non-aqueous injection solutions of the mixture ofinsulin drug-oligomer conjugates, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the composition isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The compositions may bepresented in unitdose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, an injectable, stable, sterile composition comprising amixture of insulin drug-oligomer conjugates in a unit dosage form in asealed container may be provided. The mixture of insulin drug-oligomerconjugates is provided in the form of a lyophilizate which is capable ofbeing reconstituted with a suitable pharmaceutically acceptable carrierto form a liquid composition suitable for injection thereof into asubject. The unit dosage form typically comprises from about 10 mg toabout 10 grams of the mixture of insulin drug-oligomer conjugates. Whenthe mixture of insulin drug-oligomer conjugates is substantiallywater-insoluble, a sufficient amount of emulsifying agent which isphysiologically acceptable may be employed in sufficient quantity toemulsify the mixture of insulin drug-oligomer conjugates in an aqueouscarrier. One such useful emulsifying agent is phosphatidyl choline.

[0234] Pharmaceutical compositions suitable for rectal administrationare preferably presented as unit dose suppositories. These may beprepared by admixing the mixture of insulin drug-oligomer conjugateswith one or more conventional solid carriers, for example, cocoa butter,and then shaping the resulting mixture.

[0235] Pharmaceutical compositions suitable for topical application tothe skin preferably take the form of an ointment, cream, lotion, paste,gel, spray, aerosol, or oil. Carriers which may be used includepetroleum jelly, lanoline, polyethylene glycols, alcohols, transdermalenhancers, and combinations of two or more thereof.

[0236] Pharmaceutical compositions suitable for transdermaladministration may be presented as discrete patches adapted to remain inintimate contact with the epidermis of the recipient for a prolongedperiod of time. Compositions suitable for transdermal administration mayalso be delivered by iontophoresis (see, for example, PharmaceuticalResearch 3 (6):318 (1986)) and typically take the form of an optionallybuffered aqueous solution of the mixture of insulin drug-oligomerconjugates. Suitable formulations comprise citrate or bistris buffer (pH6) or ethanol/water and contain from 0.1 to 0.2M active ingredient.

[0237] Methods of treating an insulin deficiency in a subject in need ofsuch treatment by administering an effective amount of suchpharmaceutical compositions are also provided. The effective amount ofany mixture of insulin drug-oligomer conjugates, the use of which is inthe scope of present invention, will vary somewhat from mixture tomixture, and subject to subject, and will depend upon factors such asthe age and condition of the subject and the route of delivery. Suchdosages can be determined in accordance with routine pharmacologicalprocedures known to those skilled in the art. As a general proposition,a dosage from about 0.1 to about 50 mg/kg will have therapeuticefficacy, with all weights being calculated based upon the weight of themixture of insulin drug-oligomer conjugates. Toxicity concerns at thehigher level may restrict intravenous dosages to a lower level such asup to about 10 mg/kg, with all weights being calculated based upon theweight of the active base. A dosage from about 10 mg/kg to about 50mg/kg may be employed for oral administration. Typically, a dosage fromabout 0.5 mg/kg to 5 mg/kg may be employed for intramuscular injection.The frequency of administration is usually one, two, or three times perday or as necessary to control the condition. Alternatively, thedrug-oligomer conjugates may be administered by continuous infusion. Theduration of treatment depends on the type of insulin deficiency beingtreated and may be for as long as the life of the subject.

[0238] Methods of synthesizing conjugate mixtures according toembodiments of the present invention are also provided. While thefollowing embodiments of a synthesis route are directed to synthesis ofa substantially monodispersed mixture, similar synthesis routes may beutilized for synthesizing other insulin drug-oligomer conjugate mixturesaccording to embodiments of the present invention.

[0239] A substantially monodispersed mixture of polymers comprisingpolyethylene glycol moieties is provided as illustrated in reaction 1:

[0240] R¹ is H or a lipophilic moiety. R¹ is preferably H, alkyl, arylalkyl, an aromatic moiety, a fatty acid moiety, an ester of a fatty acidmoiety, cholesteryl, or adamantyl. R¹ is more preferably H, lower alkyl,or an aromatic moiety. R¹ is most preferably H, methyl, or benzyl.

[0241] In Formula I, n is from 1 to 25. Preferably n is from 1 to 6.

[0242] X⁺ is a positive ion. Preferably X⁺ is any positive ion in acompound, such as a strong base, that is capable of ionizing a hydroxylmoiety on PEG. Examples of positive ions include, but are not limitedto, sodium ions, potassium ions, lithium ions, cesium ions, and thalliumions.

[0243] R² is H or a lipophilic moiety. R² is preferably linear orbranched alkyl, aryl alkyl, an aromatic moiety, a fatty acid moiety, oran ester of a fatty acid moiety. R²is more preferably lower alkyl,benzyl, a fatty acid moiety having 1 to 24 carbon atoms, or an ester ofa fatty acid moiety having 1 to 24 carbon atoms. R² is most preferablymethyl, a fatty acid moiety having 1 to 18 carbon atoms or an ethylester of a fatty acid moiety having 1 to 18 carbon atoms.

[0244] In Formula II, m is from 1 to 25. Preferably m is from 1 to 6.

[0245] Ms is a mesylate moiety (i.e., CH₃S(O₂)—).

[0246] As illustrated in reaction 1, a mixture of compounds having thestructure of Formula I is reacted with a mixture of compounds having thestructure of Formula II to provide a mixture of polymers comprisingpolyethylene glycol moieties and having the structure of Formula III.The mixture of compounds having the structure of Formula I is asubstantially monodispersed mixture. Preferably, at least about 96, 97,98 or 99 percent of the compounds in the mixture of compounds of FormulaI have the same molecular weight, and, more preferably, the mixture ofcompounds of Formula I is a monodispersed mixture. The mixture ofcompounds of Formula II is a substantially monodispersed mixture.Preferably, at least about 96, 97, 98 or 99 percent of the compounds inthe mixture of compounds of Formula II have the same molecular weight,and, more preferably, the mixture of compounds of Formula II is amonodispersed mixture. The mixture of compounds of Formula III is asubstantially monodispersed mixture. Preferably, at least about 96, 97,98 or 99 percent of the compounds in the mixture of compound of FormulaIII have the same molecular weight. More preferably, the mixture ofcompounds of Formula III is a monodispersed mixture.

[0247] Reaction 1 is preferably performed between about 0° C. and about40° C., is more preferably performed between about 15° C. and about 35°C., and is most preferably performed at room temperature (approximately25° C.).

[0248] Reaction 1 may be performed for various periods of time as willbe understood by those skilled in the art. Reaction 1 is preferablyperformed for a period of time between about 0.25, 0.5 or 0.75 hours andabout 2, 4 or 8 hours.

[0249] Reaction 1 is preferably carried out in an aprotic solvent suchas, but not limited to, N,N-dimethylacetamide (DMA),N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),hexamethylphosphoric triamide, tetrahydrofuran (THF), dioxane, diethylether, methyl t-butyl ether (MTBE), toluene, benzene, hexane, pentane,N-methylpyrollidinone, tetrahydronaphthalene, decahydronaphthalene,1,2-dichlorobenzene, 1,3-dimethyl-2-imidazolidinone, or a mixturethereof. More preferably, the solvent is DMF, DMA or toluene.

[0250] The molar ratio of the compound of Formula I to the compound ofFormula II is preferably greater than about 1:1. More preferably, themolar ratio is at least about 2:1. By providing an excess of thecompounds of Formula I, one can ensure that substantially all of thecompounds of Formula II are reacted, which may aid in the recovery ofthe compounds of Formula III as discussed below.

[0251] Compounds of Formula I are preferably prepared as illustrated inreaction 2:

[0252] R¹ and X⁺ are as described above and the mixture of compounds ofFormula IV is substantially monodispersed; preferably, at least about96, 97, 98 or 99 percent of the compounds in the mixture of compounds ofFormula IV have the same molecular weight; and, more preferably, themixture of compounds of Formula IV is a monodispersed mixture.

[0253] Various compounds capable of ionizing a hydroxyl moiety on thePEG moiety of the compound of Formula IV will be understood by thoseskilled in the art. The compound capable of ionizing a hydroxyl moietyis preferably a strong base. More preferably, the compound capable ofionizing a hydroxyl moiety is selected from the group consisting ofsodium hydride, potassium hydride, sodium t-butoxide, potassiumt-butoxide, butyl lithium (BuLi), and lithium diisopropylamine. Thecompound capable of ionizing a hydroxyl moiety is more preferably sodiumhydride.

[0254] The molar ratio of the compound capable of ionizing a hydroxylmoiety on the PEG moiety of the compound of Formula IV to the compoundof Formula IV is preferably at least about 1:1, and is more preferablyat least about 2:1. By providing an excess of the compound capable ofionizing the hydroxyl moiety, it is assured that substantially all ofthe compounds of Formula IV are reacted to provide the compounds ofFormula I. Thus, separation difficulties, which may occur if bothcompounds of Formula IV and compounds of Formula I were present in thereaction product mixture, may be avoided.

[0255] Reaction 2 is preferably performed between about 0° C. and about40° C., is more preferably performed between about 0° C. and about 35°C., and is most preferably performed between about 0° C. and roomtemperature (approximately 25° C.).

[0256] Reaction 2 may be performed for various periods of time as willbe understood by those skilled in the art. Reaction 2 is preferablyperformed for a period of time between about 0.25, 0.5 or 0.75 hours andabout 2, 4 or 8 hours.

[0257] Reaction 2 is preferably carried out in an aprotic solvent suchas, but not limited to, N,N-dimethylacetamide (DMA),N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),hexamethylphosphoric triamide, tetrahydrofuran (THF), dioxane, diethylether, methyl t-butyl ether (MTBE), toluene, benzene, hexane, pentane,N-methylpyrollidinone, dichloromethane, chloroform,tetrahydronaphthalene, decahydronaphthalene, 1,2-dichlorobenzene,1,3-dimethyl-2-imidazolidinone, or a mixture thereof. More preferably,the solvent is DMF, dichloromethane or toluene.

[0258] Compounds of Formula II are preferably prepared as illustrated inreaction 3:

[0259] R² and Ms are as described above and the compound of Formula V ispresent as a substantially monodispersed mixture of compounds of FormulaV; preferably at least about 96, 97, 98 or 99 percent of thecompounds-in the mixture of compounds of Formula V have the samemolecular weight; and, more preferably, the mixture of compounds ofFormula V is a monodispersed mixture.

[0260] Q is a halide, preferably chloride or fluoride.

[0261] CH₃S(O₂)Q is methanesulfonyl halide. The methanesulfonyl halideis preferably methanesulfonyl chloride or methanesulfonyl fluoride. Morepreferably, the methanesulfonyl halide is methanesulfonyl chloride.

[0262] The molar ratio of the methane sulfonyl halide to the compound ofFormula V is preferably greater than about 1:1, and is more preferablyat least about 2:1. By providing an excess of the methane sulfonylhalide, it is assured that substantially all of the compounds of FormulaV are reacted to provide the compounds of Formula II. Thus, separationdifficulties, which may occur if both compounds of Formula V andcompounds of Formula II were present in the reaction product mixture,may be avoided.

[0263] Reaction 3 is preferably performed between about −10° C. andabout 40° C., is more preferably performed between about 0C and about35° C., and is most preferably performed between about 0C and roomtemperature (approximately 25° C.).

[0264] Reaction 3 may be performed for various periods of time as willbe understood by those skilled in the art. Reaction 3 is preferablyperformed for a period of time between about 0.25, 0.5 or 0.75 hours andabout 2, 4 or 8 hours.

[0265] Reaction 3 is preferably carried out in the presence of analiphatic amine including, but not limited to, monomethylamine,dimethylamine, trimethylamine, monoethylamine, diethylamine,triethylamine, monoisopropylamine, diisopropylamine, mono-n-butylamine,di-n-butylamine, tri-n-butylamine, monocyclohexylamine,dicyclohexylamine, or mixtures thereof. More preferably, the aliphaticamine is a tertiary amine such as triethylamine.

[0266] As will be understood by those skilled in the art, varioussubstantially monodispersed mixtures of compounds of Formula V arecommercially available. For example, when R² is H or methyl, thecompounds of Formula V are PEG or mPEG compounds, respectively, whichare commercially available from Aldrich of Milwaukee, Wis.; Fluka ofSwitzerland, and/or TCl America of Portland, Oreg.

[0267] When R² is a lipophilic moiety such as, for example, higheralkyl, fatty acid, an ester of a fatty acid, cholesteryl, or adamantyl,the compounds of Formula V may be provided by various methods as will beunderstood by those skilled in the art. The compounds of Formula V arepreferably provided as follows:

[0268] R² is a lipophilic moiety, preferably higher alkyl, fatty acidester, cholesteryl, or adamantyl, more preferably a lower alkyl ester ofa fatty acid, and most preferably an ethyl ester of a fatty acid havingfrom 1 to 18 carbon atoms.

[0269] R³ is H, benzyl, trityl, tetrahydropyran, or other alcoholprotecting groups as will be understood by those skilled in the art.

[0270] X₂ ⁺ is a positive ion as described above with respect to X⁺.

[0271] The value of m is as described above.

[0272] Regarding reaction 4, a mixture of compounds of Formula VI isreacted with a mixture of compounds of Formula VII under reactionconditions similar to those described above with reference to reaction1. The mixture of compounds of Formula VI is a substantiallymonodispersed mixture. Preferably, at least about 96, 97, 98 or 99percent of the compounds in the mixture of compounds of Formula VI havethe same molecular weight. More preferably, the mixture of compounds ofFormula VI is a monodispersed mixture. The mixture of compounds ofFormula VII is a substantially monodispersed mixture. Preferably, atleast about 96, 97, 98 or 99 percent of the compounds in the mixture ofcompounds of Formula VII have the same molecular weight. Morepreferably, the mixture of compounds of Formula VII is a monodispersedmixture.

[0273] Regarding reaction 5, the compound of Formula VIII may behydrolyzed to convert the R³ moiety into an alcohol by various methodsas will be understood by those skilled in the art. When R³ is benzyl ortrityl, the hydrolysis is preferably performed utilizing H₂ in thepresence of a palladium-charcoal catalyst as is known by those skilledin the art. Of course, when R³ is H, reaction 5 is unnecessary.

[0274] The compound of Formula VI may be commercially available or beprovided as described above with reference to reaction 3. The compoundof Formula VII may be provided as described above with reference toreaction 2.

[0275] Substantially monodispersed mixtures of polymers comprising PEGmoieties and having the structure of Formula III above can further bereacted with other substantially monodispersed polymers comprising PEGmoieties in order to extend the PEG chain. For example, the followingscheme may be employed:

[0276] Ms, m and n are as described above with reference to reaction 1;p is similar to n and m, and X₂ ⁺ is similar to X⁺ as described abovewith reference to reaction 1. Q is as described above with reference toreaction 3. R² is as described above with reference to reaction 1 and ispreferably lower alkyl. R¹ is H. Reaction 6 is preferably performed in amanner similar to that described above with reference to reaction 3.Reaction 7 is preferably performed in a manner similar to that describedabove with reference to reaction 1. Preferably, at least about 96, 97,98 or 99 percent of the compounds in the mixture of compounds of FormulaIII have the same molecular weight, and, more preferably, the mixture ofcompounds of Formula III is a monodispersed mixture. The mixture ofcompounds of Formula X is a substantially monodispersed mixture.Preferably, at least about 96, 97, 98 or 99 percent of the compounds inthe mixture of compounds of Formula X have the same molecular weight,and, more preferably, the mixture of compounds of Formula X is amonodispersed mixture.

[0277] A process according to embodiments of the present invention isillustrated by the scheme shown in FIG. 1, which will now be described.The synthesis of substantially monodispersed polyethyleneglycol-containing oligomers begins by the preparation of the monobenzylether (1) of a substantially monodispersed polyethylene glycol. Anexcess of a commercially available substantially monodispersedpolyethylene glycol is reacted with benzyl chloride in the presence ofaqueous sodium hydroxide as described by Coudert et al (SyntheticCommunications, 16(1): 19-26 (1986)). The sodium salt of 1 is thenprepared by the addition of NaH, and this sodium salt is allowed toreact with the mesylate synthesized from the ester of a hydroxyalkanoicacid (2). The product (3) of the displacement of the mesylate isdebenzylated via catalytic hydrogenation to obtain the alcohol (4). Themesylate (5) of this alcohol may be prepared by addition ofmethanesulfonyl chloride and used as the electrophile in the reactionwith the sodium salt of the monomethyl ether of a substantiallymonodispersed polyethylene glycol derivative, thereby extending thepolyethylene glycol portion of the oligomer to the desired length,obtaining the elongated ester (6). The ester may be hydrolyzed to theacid (7) in aqueous base and transformed into the activated ester (8) byreaction with a carbodiimide and N-hydroxysuccinimide. While theoligomer illustrated in FIG. 1 is activated using N-hydroxysuccinimide,it is to be understood that various other reagents may be used toactivate oligomers of the present invention including, but not limitedto, active phenyl chloroformates such as para-nitrophenyl chloroformate,phenyl chloroformate, 3,4-phenyldichloroformate, and3,4-phenyldichloroformate;. tresylation; and acetal formation.

[0278] Still referring to FIG. 1, q is from 1 to 24. Preferably, q isfrom 1 to 18, and q is more preferably from 4 to 16. R⁴ is a moietycapable of undergoing hydrolysis to provide the carboxylic acid. R⁴ ispreferably lower alkyl and is more preferably ethyl. The variables n andm are as described above with reference to reaction 1.

[0279] All starting materials used in the procedures described hereinare either commercially available or can be prepared by methods known inthe art using commercially available starting materials.

[0280] The present invention will now be described with reference to thefollowing examples. It should be appreciated that these examples are forthe purposes of illustrating aspects of the present invention, and donot limit the scope of the invention as defined by the claims.

EXAMPLES Examples 1 Through 10

[0281] Reactions in Examples 1 through 10 were carried out undernitrogen with magnetic stirring, unless otherwise specified. “Work-up”denotes extraction with an organic solvent, washing of the organic phasewith saturated NaCl solution, drying (MgSO₄), and evaporation (rotaryevaporator). Thin layer chromatography was conducted with Merck glassplates precoated with silica gel 60° F.-254 and spots were visualized byiodine vapor. All mass spectra were determined by MacromolecularResources Colorado State University, Colo. and are reported in the orderm/z, (relative intensity). Elemental analyses and melting points wereperformed by Galbraith Laboratories, Inc., Knoxville, Tenn. Examples1-10 refer to the scheme illustrated in FIG. 2.

Example 1 8-Methoxy-1-(methylsulfonyl)oxy-3,6-dioxaoctane (9)

[0282] A solution of non-polydispersed triethylene glycol monomethylether molecules (4.00 mL, 4.19 g, 25.5 mmol) and triethylamine (4.26 mL,3.09 g, 30.6 mmol) in dry dichloromethane (50 mL) was chilled in an icebath and place under a nitrogen atmosphere. A solution ofmethanesulfonyl chloride (2.37 mL, 3.51 g, 30.6 mmol) in drydichloromethane (20 mL) was added dropwise from an addition funnel. Tenminutes after the completion of the chloride addition, the reactionmixture was removed from the ice bath and allowed to come to roomtemperature. The mixture was stirred for an additional hour, at whichtime TLC (CHCl₃ with 15% MeOH as the elutant) showed no remainingtriethylene glycol monomethyl ether.

[0283] The reaction mixture was diluted with another 75 mL ofdichloromethane and washed successively with saturated NaHCO₃, water andbrine. The organics were dried over Na₂SO₄, filtered and concentrated invacuo to give a non-polydispersed mixture of compounds 9 as a clear oil(5.3 1g, 86%).

Example 2 Ethylene glycol mono methyl ether (10) (m=4,5,6)

[0284] To a stirred solution of non-polydispersed compound 11 (35.7mmol) in dry DMF (25.7 mL), under N₂ was added in portion a 60%dispersion of NaH in mineral oil, and the mixture was stirred at roomtemperature for 1 hour. To this salt 12 was added a solution ofnon-polydispersed mesylate 9 (23.36) in dry DMF (4 ml) in a singleportion, and the mixture was stirred at room temperature for 3.5 hours.Progress of the reaction was monitored by TLC (12% CH₃OH—CHCl₃). Thereaction mixture was diluted with an equal amount of 1N HCl, andextracted with ethyl acetate (2×20 ml) and discarded. Extraction ofaqueous solution and work-up gave non-polydispersed polymer 10 (82-84%yield).

Example 3 3,6,9,12,15,18,21-Heptaoxadocosanol (10) (m=4)

[0285] Oil; Rf 0.46 (methanol:chloroform=3:22); MS m/z calc'd forC₁₅H₃₂O₈ 340.21 (M⁺+1), found 341.2.

Example 4 3,6,9,12,15,18,21,24-Octaoxapentacosanol (10) (m=5)

[0286] Oil; Rf 0.43 (methanol:chloroform=6:10); MS m/z calc'd for C₁₇H₃₆O₉ 384.24 (M⁺+1), found 385.3.

Example 5 3,6,9,12,15,18,21,24,27-Nonaoxaoctacosanol (10) (m=6)

[0287] Oil; Rf 0.42 (methanol:chloroform=6:10); MS m/z calc'd forC₁₉H₄₀O₁₀ 428.26 (M⁺+1), found 429.3.

Example 620-methoxy-1-(methylsulfonyl)oxy-3,6,9,12,15,18-hexaoxaeicosane (14)

[0288] Non-polydispersed compound 14 was obtained in quantitative yieldfrom the alcohol 13 (m=4) and methanesulfonyl chloride as described for9, as an oil; Rf 0.4 (ethyl acetate:acetonitrile=1:5); MS m/z calc'd forC₁₇H₃₇O₁₀ 433.21 (M⁺+1), found 433.469.

Example 7 Ethylene glycol mono methyl ether (15) (m=3,4,5)

[0289] The non-polydispersed compounds 15 were prepared from a diol byusing the procedure described above for compound 10.

Example 8 3,6,9,12,15,18,21,24,27,30-Decaoxaheneicosanol (15) (m=3)

[0290] Oil; Rf 0.41 (methanol:chloroform=6:10); MS m/z calc'd forC₂₁H4₄O₁₁ 472.29 (M⁺+1), found 472.29.

Example 9 3,6,9,12,15,18,21,24,27,30,33-Unecaoxatetratricosanol (15)(m=4)

[0291] Oil; Rf 0.41 (methanol:chloroform=6:10); MS m/z calc'd forC₂₃H₄₈O₁₂ 516.31 (M⁺+1), found 516.31.

Example 10 3,6,9,12,15,18,21,24,27,30,33,36-Dodecaoxaheptatricosanol(15) (m=5)

[0292] Oil; Rf 0.41 (methanol:chloroform=6: 10); MS m/z calc'd forC₂₅H₅₂O₁₃ 560.67 (M⁺+1), found 560.67.

[0293] Examples 11 through 18 refer to the scheme illustrated in FIG. 3.

Example 11 Hexaethylene glycol monobenzyl ether (16)

[0294] An aqueous sodium hydroxide solution prepared by dissolving 3.99g (100 mmol) NaOH in 4 ml water was added slowly to non-polydispersedhexaethylene glycol (28.175 g, 25 ml, 100 mmol). Benzyl chloride (3.9 g,30.8 mmol, 3.54 ml) was added and the reaction mixture was heated withstirring to 100° C. for 18 hours. The reaction mixture was then cooled,diluted with brine (250 ml) and extracted with methylene chloride (200ml×2). The combined organic layers were washed with brine once, driedover Na₂SO₄, filtered and concentrated in vacuo to a dark brown oil. Thecrude product mixture was purified via flash chromatography (silica gel,gradient elution: ethyl acetate to 9/1 ethyl acetate/methanol) to yield8.099 g (70%) of non-polydispersed 16 as a yellow oil.

Example 12 Ethyl 6-methylsulfonyloxyhexanoate (17)

[0295] A solution of non-polydispersed ethyl 6-hydroxyhexanoate (50.76ml, 50.41 g, 227 mmol) in dry dichloromethane (75 ml) was chilled in aice bath and placed under a nitrogen atmosphere. Triethylamine (34.43ml, 24.99 g, 247 mmol) was added. A solution of methanesulfonyl chloride(19.15 ml, 28.3 g, 247 mmol) in dry dichloromethane (75 ml) was addeddropwise from an addition funnel. The mixture was stirred for three andone half hours, slowly being allowed to come to room temperature as theice bath melted. The mixture was filtered through silica gel, and thefiltrate was washed successively with water, saturated NaHCO₃, water andbrine. The organics were dried over Na₂SO₄, filtered and concentrated invacuo to a pale yellow oil. Final purification of the crude product wasachieved by flash chromatography (silica gel, 1/1 hexanes/ethyl acetate)to give the non-polydispersed product (46.13 g, 85%) as a clear,colorless oil. FAB MS: m/e 239 (M+H), 193 (M−C₂H₅O).

Example 136-{2-[2-(2-{2-[2-(2-Benzyloxyethoxy)ethoxylethoxy}-ethoxy)-ethoxyl-ethoxy}-hexanoicacid ethyl ester (18)

[0296] Sodium hydride (3.225 g or a 60 % oil dispersion, 80.6 mmol) wassuspended in 80 ml of anhydrous toluene, placed under a nitrogenatmosphere and cooled in an ice bath. A solution of thenon-polydispersed alcohol 16 (27.3 g, 73.3 mmol) in 80 ml dry toluenewas added to the NaH suspension. The mixture was stirred at 0° C. forthirty minutes, allowed to come to room temperature and stirred foranother five hours, during which time the mixture became a clear brownsolution. The non-polydispersed mesylate 17 (19.21 g, 80.6 mmol) in 80ml dry toluene was added to the NaH/alcohol mixture, and the combinedsolutions were stirred at room temperature for three days. The reactionmixture was quenched with 50 ml methanol and filtered through basicalumina. The filtrate was concentrated in vacuo and purified by flashchromatography (silica gel, gradient elution: 3/1 ethyl acetate/hexanesto ethyl acetate) to yield the non-polydispersed product as a paleyellow oil (16.52 g, 44%). FAB MS: m/e 515 (M+H).

Example 146-{2-[2-(2-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}-ethoxy)-ethoxyl-ethoxy}-hexanoicacid ethyl ester (19)

[0297] Non-polydispersed benzyl ether 18 (1.03 g, 2.0 mmol) wasdissolved in 25 ml ethanol. To this solution was added 270 mg 10 % Pd/C,and the mixture was placed under a hydrogen atmosphere and stirred forfour hours, at which time TLC showed the complete disappearance of thestarting material. The reaction mixture was filtered through Celite 545to remove the catalyst, and the filtrate was concentrated in vacuo toyield the non-polydispersed title compound as a clear oil (0.67 g, 79%). FAB MS: m/e 425 (M+H), 447 (M+Na).

Example 156-{2-[2-(2-{2-[2-(2-methylsulfonylethoxy)ethoxy]ethoxy}-ethoxy)-ethoxy]-ethoxy}-hexanoicacid ethyl ester (20)

[0298] The non-polydispersed alcohol 19 (0.835 g, 1.97 mmol) wasdissolved in 3.5 ml dry dichloromethane and placed under a nitrogenatmosphere. Triethylamine (0.301 ml, 0.219 g, 2.16 mmol) was added andthe mixture was chilled in an ice bath. After two minutes, themethanesulfonyl chloride (0.16 ml, 0.248 g, 2.16 mmol) was added. Themixture was stirred for 15 minutes at 0° C., then at room temperaturefor two hours. The reaction mixture was filtered through silica gel toremove the triethylammonium chloride, and the filtrate was washedsuccessively with water, saturated NaHCO₃, water and brine. The organicswere dried over Na₂SO₄, filtered and concentrated in vacuo. The residuewas purified by column chromatography (silica gel, 9/1 ethylacetate/methanol) to give non-polydispersed compound 20 as a clear oil(0.819 g, 83%). FAB MS: m/e 503 (M+H).

Example 166-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-hexanoicacid ethyl ester (21)

[0299] NaH (88 mg of a 60% dispersion in oil, 2.2 mmol) was suspended inanhydrous toluene (3 ml) under N₂ and chilled to 0° C. Non-polydisperseddiethylene glycol monomethyl ether (0.26 ml, 0.26 g, 2.2 mmol) that hadbeen dried via azeotropic distillation with toluene was added. Thereaction mixture was allowed to warm to room temperature and stirred forfour hours, during which time the cloudy grey suspension became clearand yellow and then turned brown. Mesylate 20 (0.50 g, 1.0 mmol) in 2.5ml dry toluene was added. After stirring at room temperature over night,the reaction was quenched by the addition of 2 ml of methanol and theresultant solution was filtered through silica gel. The filtrate wasconcentrated in vacuo and the FAB MS: m/e 499 (M+H), 521 (M+Na).Additional purification by preparatory chromatography (silica gel, 19/3chloroform/methanol) provided the non-polydispersed product as a clearyellow oil (0.302 g 57 %). FAB MS: m/e 527 (M+H), 549 (M+Na).

Example 176-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-hexanoicacid (22)

[0300] Non-polydispersed ester 21 (0.25 g, 0.46 mmol) was stirred for 18hours in 0.71 ml of 1 N NaOH. After 18 hours, the mixture wasconcentrated in vacuo to remove the alcohol and the residue dissolved ina further 10 ml of water. The aqueous solution was acidified to pH 2with 2 N HCl and the product was extracted into dichloromethane (30ml×2). The combined organics were then washed with brine (25 ml×2),dried over Na₂SO₄, filtered and concentrated in vacuo to yield thenon-polydispersed title compound as a yellow oil (0.147 g, 62%). FAB MS:m/e 499 (M+H), 521 (M+Na).

Example 186-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxyl-ethoxy}-ethoxy)-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (23)

[0301] Non-polydispersed acid 22 (0.209 g, 0.42 mmol) were dissolved in4 ml of dry dichloromethane and added to a dry flask already containingNHS (N-hydroxysuccinimide) (57.8 mg, 0.502 mmol) and EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (98.0 mg,0.502 mmol) under a N₂ atmosphere. The solution was stirred at roomtemperature overnight and filtered through silica gel to remove excessreagents and the urea formed from the EDC. The filtrate was concentratedin vacuo to provide the non-polydispersed product as a dark yellow oil(0.235 g, 94%). FAB MS: m/e 596 (M+H), 618 (M+Na).

[0302] Examples 19 through 24 refer to the scheme illustrated in FIG. 4.

Example 19 Mesylate of triethylene glycol monomethyl ether (24)

[0303] To a solution of CH₂Cl₂ (100 mL) cooled to 0° C. in an ice bathwas added non-polydispersed triethylene glycol monomethyl ether (25 g,0.15 mol). Then triethylamine (29.5 mL, 0.22 mol) was added and thesolution was stirred for 15 min at 0° C., which was followed by dropwiseaddition of methanesulfonyl chloride (13.8 mL, 0.18 mol, dissolved in 20mL CH₂Cl₂). The reaction mixture was stirred for 30 min at 0° C.,allowed to warm to room temperature, and then stirred for 2 h. The crudereaction mixture was filtered through Celite (washed CH₂Cl₂˜200 mL),then washed with H₂O (300 mL), 5% NaHCO₃ (300 mL), H₂O (300 mL), sat.NaCl (300 mL), dried MgSO₄, and evaporated to dryness. The oil was thenplaced on a vacuum line for ˜2 h to ensure dryness and afforded thenon-polydispersed title compound as a yellow oil (29.15 g, 80% yield).

Example 20 Heptaethylene glycol inonomethyl ether (25)

[0304] To a solution of non-polydispersed tetraethylene glycol (51.5 g,0.27 mol) in THF (1 L) was added potassium t-butoxide (14.8 g, 0.13 mol,small portions over ˜30 min). The reaction mixture was then stirred for1 h and then 24 (29.15 g, 0.12 mol) dissolved in THF (90 mL) was addeddropwise and the reaction mixture was stirred overnight. The crudereaction mixture was filtered through Celite (washed CH₂Cl₂, ˜200 mL)and evaporated to dryness. The oil was then dissolved in HCl (250 mL, 1N) and washed with ethyl acetate (250 mL) to remove excess 24.Additional washings of ethyl acetate (125 mL) may be required to removeremaining 24. The aqueous phase was washed repetitively with CH₂Cl₂ (125mL volumes) until most of the 25 has been removed from the aqueousphase. The first extraction will contain 24, 25, and dicoupled sideproduct and should be back extracted with HCl (125 mL, 1N). The organiclayers were combined and evaporated to dryness. The resultant oil wasthen dissolved in CH₂Cl₂ (100 mL) and washed repetitively with H₂O (50mL volumes) until 25 was removed. The aqueous fractions were combined,total volume 500 mL, and NaCl was added until the solution became cloudyand then was washed with CH₂Cl₂ (2×500 mL). The organic layers werecombined, dried MgSO₄, and evaporated to dryness to afford a thenon-polydispersed title compound as an oil (16.9 g, 41% yield). It maybe desirable to repeat one or more steps of the purification procedureto ensure high purity.

Example 21 8-Bromooctoanate (26)

[0305] To a solution of 8-bromooctanoic acid (5.0 g, 22 mmol) in ethanol(100 mL) was added H₂SO₄ (0.36 mL, 7.5 mmol) and the reaction was heatedto reflux with stirring for 3 h. The crude reaction mixture was cooledto room temperature and washed H₂O (100 mL), sat. NaHCO₃ (2×100 mL), H₂O(100 mL), dried MgSO₄, and evaporated to dryness to afford a clear oil(5.5 g, 98% yield).

Example 22 Synthesis of MPEG7-C8 ester (27)

[0306] To a solution of the non-polydispersed compound 25 (3.0 g, 8.8mmol) in ether (90 mL) was added potassium t-butoxide (1.2 g, 9.6 mmol)and the reaction mixture was stirred for 1 h. Then dropwise addition ofthe non-polydispersed compound 26 (2.4 g, 9.6 mmol), dissolved in ether(10 mL), was added and the reaction mixture was stirred overnight. Thecrude reaction mixture was filtered through Celite (washed CH₂Cl₂, ˜200mL) and evaporated to dryness. The resultant oil was dissolved in ethylacetate and washed H₂O (2×200 mL), dried MgSO₄, and evaporated todryness. Column chromatography (Silica, ethyl acetate to ethylacetate/methanol, 10:1) was performed and afforded the non-polydispersedtitle compound as a clear oil (0.843 g, 19% yield).

Example 23 MPEG7-C8 acid (28)

[0307] To the oil of the non-polydispersed compound 27 (0.70 g, 1.4mmol) was added 1N NaOH (2.0 mL) and the reaction mixture was stirredfor 4 h. The crude reaction mixture was concentrated, acidified (pH˜2),saturated with NaCl, and washed CH₂Cl₂ (2×50 mL). The organic layerswere combined, washed sat. NaCl, dried MgSO₄, and evaporated to drynessto afford the non-polydispersed title compound as a clear oil (0.35 g,53% yield).

Example 24 Activation of MPEG7-C8 acid (29)

[0308] Non-polydispersed mPEG7-C8-acid 28 0.31 g, 0.64 mmol) wasdissolved in 3 ml of anhydrous methylene chloride and then solution ofN-hydroxysuccinimide (0.079 g, 0.69 mmol) and EDCI.HCl (135.6.mg, 0.71mmol) in anhydrous methylene chloride added. Reaction was stirred forseveral hours, then washed with 1N HCl, water, dried over MgSO₄,filtered and concentrated. Crude material was purified by columnchromatography, concentrated to afford the non-polydispersed titlecompound as a clear oil and dried via vacuum.

[0309] Examples 25 through 29 refer to the scheme illustrated in FIG. 5.

Example 25 10-hydroxydecanoate (30)

[0310] To a solution of non-polydispersed 10-hydroxydecanoic acid (5.0g, 26.5 mmol) in ethanol (100 mL) was added H₂S0₄ (0.43 mL, 8.8 mmol)and the reaction was heated to reflux with stirring for 3 h. The crudereaction mixture was cooled to room temperature and washed H₂O (100 mL),sat. NaHCO₃ (2×100 mL), H₂0 (100 mL), dried MgSO₄, and evaporated todryness to afford the non-polydispersed title compound as a clear oil(6.9 g, 98% yield).

Example 26 Mesylate of 10-hydroxydecanoate (31)

[0311] To a solution of CH₂Cl₂ (27 mL) was added non-polydispersed10-hydroxydecanoate 30 (5.6 g, 26 mmol) and cooled to 0° C. in an icebath. Then triethylamine (5 mL, 37 mmol) was added and the reactionmixture was stirred for 15 min at 0° C. Then methanesulfonyl chloride(2.7 mL, 24 mmol) dissolved in CH₂Cl₂ (3 mL) was added and the reactionmixture was stirred at 0° C. for 30 min, the ice bath was removed andthe reaction was stirred for an additional 2 h at room temperature. Thecrude reaction mixture was filtered through Celite (washed CH₂Cl₂, 80mL) and the filtrate was washed H₂O (100 mL), 5% NaHCO₃ (2×100 mL), H₂O(100 mL), sat. NaCl (100 mL), dried MgSO₄, and evaporated to dryness toafford the non-polydispersed title compound as a yellowish oil (7.42 g,97% yield).

Example 27 MPEG₇-C₁₀Ester (32)

[0312] To a solution of non-polydispersed heptaethylene glycolmonomethyl ether 25 (2.5 g, 7.3 mmol) in tetrahydrofuran (100 mL) wasadded sodium hydride (0.194 g, 8.1 mmol) and the reaction mixture wasstirred for 1 h. Then dropwise addition of mesylate of non-polydispersed10-hydroxydecanoate 31 (2.4 g, 8.1 mmol), dissolved in tetrahydrofuran(10 mL), was added and the reaction mixture was stirred overnight. Thecrude reaction mixture was filtered through Celite (washed CH₂Cl₂, ˜200mL) and evaporated to dryness. The resultant oil was dissolved in ethylacetate and washed H₂O (2×200 mL), dried MgSO₄, evaporated to dryness,chromatographed (silica, ethyl acetate/methanol, 10:1), andchromatographed (silica, ethyl acetate) to afford the non-polydispersedtitle compound as a clear oil (0.570 g, 15% yield).

Example 28 MPEG₇-C₁₀ Acid (33)

[0313] To the oil of non-polydispersed mPEG₇-C₁₀ ester 32 (0.570 g, 1.1mmol) was added 1N NaOH (1.6 mL) and the reaction mixture was stirredovernight. The crude reaction mixture was concentrated, acidified(pH˜2), saturated with NaCl, and washed CH₂Cl₂ (2×50 mL). The organiclayers were combined, washed sat. NaCl (2×50 mL), dried MgSO₄, andevaporated to dryness to afford the non-polydispersed title compound asa clear oil (0.340 g, 62% yield).

Example 29 Activation of MPEG₇-C₁₀ Acid (34)

[0314] The non-polydispersed acid 33 was activated using proceduressimilar to those described above in Example 24.

[0315] Examples 30 and 31 refer to the scheme illustrated in FIG. 6.

Example 30 Synthesis of C18(PEG6) Oligomer (36)

[0316] Non-polydispersed stearoyl chloride 35 (0.7g, 2.31 mmol) wasadded slowly to a mixture of PEG6 (5 g, 17.7 mmol) and pyridine (0.97g,12.4 mmol) in benzene. The reaction mixture was stirred for severalhours (˜5). The reaction was followed by TLC using ethylacetate/methanolas a developing solvent. Then the reaction mixture was washed withwater, dried over MgSO₄, concentrated and dried via vacuum. Purifiednon-polydispersed compound 36 was analyzed by FABMS: m/e 549/ M⁺H.

Example 31 Activation of C18(PEG6) Oligomer

[0317] Activation of non-polydispersed C18(PEG6) oligomer wasaccomplished in two steps:

[0318] 1) Non-polydispersed stearoyl-PEG6 36 (0.8 g, 1.46 mmol ) wasdissolved in toluene and added to a phosgene solution (10 ml, 20% intoluene) which was cooled with an ice bath. The reaction mixture wasstirred for 1 h at 0° C. and then for 3 h at room temperature. Thenphosgene and toluene were distilled off and the remainingnon-polydispersed stearoyl PEG6 chloroformate 37 was dried over P₂O₅overnight.

[0319] 2) To a solution of non-polydispersed stearoyl PEG6 chloroformate36 (0.78g, 1.27 mmol) and TEA (128 mg, 1.27 mmol) in anhydrous methylenechloride, N-hydroxy succinimide (NHS) solution in methylene chloride wasadded. The reaction mixture was stirred for 16 hours, then washed withwater, dried over MgSO₄, filtered, concentrated and dried via vacuum toprovide the non-polydispersed activated C 18(PEG6) oligomer 38.

[0320] Examples 32 through 37 refer to the scheme illustrated in FIG. 7.

Example 32 Tetraethylene glycol monobenzylether (39)

[0321] To the oil of non-polydispersed tetraethylene glycol (19.4 g,0.10 mol) was added a solution of NaOH (4.0 g in 4.0 mL) and thereaction was stirred for 15 mm. Then benzyl chloride (3.54 mL, 30.8mmol) was added and the reaction mixture was heated to 100° C. andstirred overnight. The reaction mixture was cooled to room temperature,diluted with sat. NaCl (250 mL), and washed CH₂Cl₂ (2×200 mL). Theorganic layers were combined, washed sat. NaCl, dried MgSO₄, andchromatographed (silica, ethyl acetate) to afford the non-polydispersedtitle compound as a yellow oil (6.21 g, 71 % yield).

Example 33 Mesylate of tetraethylene glycol monobenzylether (40)

[0322] To a solution of CH₂Cl₂ (20 mL) was added non-polydispersedtetraethylene glycol monobenzylether 39 (6.21 g, 22 mmol) and cooled to0° C. in an ice bath. Then triethylamine (3.2 mL, 24 mmol) was added andthe reaction mixture was stirred for 15 min at 0° C. Thenmethanesulfonyl chloride (1.7 mL, 24 mmol) dissolved in CH₂Cl₂ (2 mL)was added and the reaction mixture was stirred at 0° C. for 30 min, theice bath was removed and the reaction was stirred for an additional 2 hat room temperature. The crude reaction mixture was filtered throughCelite (washed CH₂Cl₂, 80 mL) and the filtrate was washed H₂O (100 mL),5% NaHCO₃ (2×100 mL), H₂0 (100 mL), sat. NaCl (100 mL), and dried MgSO₄.The resulting yellow oil was chromatographed on a pad of silicacontaining activated carbon (10 g) to afford the non-polydispersed titlecompound as a clear oil (7.10 g, 89% yield).

Example 34 Octaethylene glycol monobenzylether (41)

[0323] To a solution of tetrahydrofuran (140 mL) containing sodiumhydride (0.43 g, 18 mmol) was added dropwise a solution ofnon-polydispersed tetraethylene glycol (3.5 g, 18 mmol) intetrahydrofuran (I0 mL) and the reaction mixture was stirred for 1 h.Then mesylate of non-polydispersed tetraethylene glycol monobenzylether40 (6.0 g, 16.5 mmol) dissolved in tetrahydrofuran (10 mL) was addeddropwise and the reaction mixture was stirred overnight. The crudereaction mixture was filtered through Celite (washed, CH₂Cl₂, 250 mL)and the filtrate was washed H₂O, dried MgSO₄, and evaporated to dryness.The resultant oil was chromatographed (silica, ethyl acetate/methanol,10:1) and chromatographed (silica, chloroform/methanol, 25:1) to affordthe non-polydispersed title compound as a clear oil (2.62 g, 34% yield).

Example 35 Synthesis of Stearate PEG8-Benzyl (43)

[0324] To a stirred cooled solution of non-polydispersed octaethyleneglycol monobenzylether 41 (0.998 g, 2.07 mmol) and pyridine (163.9 mg,2.07 mmol) was added non-polydispersed stearoyl chloride 42 (627.7 mg,2.07 mmol) in benzene. The reaction mixture was stirred overnight (18hours). The next day the reaction mixture was washed with water, driedover MgSO₄, concentrated and dried via vacuum. Then the crude productwas chromatographed on flash silica gel column, using 10% methanol/90%chloroform. The fractions containing the product were combined,concentrated and dried via vacuum to afford the non-polydispersed titlecompound.

Example 36 Hydrogenolysis of Stearate-PEG8-Benzyl

[0325] To a methanol solution of non-polydispersed stearate-PEG8-Bzl 43(0.854 g 1.138 mmol ) Pd/C(10%) (palladium, 10% wt. on activated carbon)was added. The reaction mixture was stirred overnight (18 hours) underhydrogen. Then the solution was filtered, concentrated and purified byflash column chromatography using 10% methanol/90% chloroform, fractionswith R_(t)=0.6 collected, concentrated and dried to provide thenon-polydispersed acid 44.

Example 37 Activation of C18(PEG8) Oligomer

[0326] Two step activation of non-polydispersed stearate-PEG8 oligomerwas performed as described for stearate-PEG6 in Example 31 above toprovide the non-polydispersed activated C18(PEG8) oligomer 45.

Example 38 Synthesis of Activated Triethylene Glycol MonomethylOligomers

[0327] The following description refers to the scheme illustrated inFIG. 8. A solution of toluene containing 20% phosgene (100 ml,approximately 18.7 g, 189 mmol phosgene) was chilled to 0° C. under a N₂atmosphere. Non-polydispersed mTEG (triethylene glycol, monomethylether, 7.8 g, 47.5 mmol) was dissolved in 25 mL anhydrous ethyl acetateand added to the chilled phosgene solution. The mixture was stirred forone hour at 0° C., then allowed to warm to room temperature and stirredfor another two and one half hours. The remaining phosgene, ethylacetate and toluene were removed via vacuum distillation to leave thenon-polydispersed mTEG chloroformate 46 as a clear oily residue.

[0328] The non-polydispersed residue 46 was dissolved in 50 mL of drydichloromethane to which was added TEA (triethyleamine, 6.62 mL, 47.5mmol) and NHS (N-hydroxysuccinimide, 5.8 g, 50.4 mmol). The mixture wasstirred at room temperature under a dry atmosphere for twenty hoursduring which time a large amount of white precipitate appeared. Themixture was filtered to remove this precipitate and concentrated invacuo. The resultant oil 47 was taken up in dichloromethane and washedtwice with cold deionized water, twice with 1N HCl and once with brine.The-organics were dried over MgSO₄, filtered and concentrated to providethe non-polydispersed title compound as a clear, light yellow oil. Ifnecessary, the NHS ester could be further purified by flashchromatography on silica gel using EtOAc as the elutant.

Example 39 Synthesis of Activated Palmitate-TEG Oligomers

[0329] The following description refers to the scheme illustrated inFIG. 9. Non-polydispersed palmitic anhydride (5 g; 10 mmol) wasdissolved in dry THF (20 mL) and stirred at room temperature. To thestirring solution, 3 mol excess of pyridine was added followed bynon-polydispersed triethylene glycol (1.4 mL). The reaction mixture wasstirred for 1 hour (progress of the reaction was monitored by TLC; ethylacetate-chloroform; 3:7). At the end of the reaction, THF was removedand the product was mixed with 10% H₂SO₄ acid and extracted ethylacetate (3×30 mL). The combined extract was washed sequentially withwater, brine, dried over MgSO₄, and evaporated to give non-polydispersedproduct 48. A solution of N,N′-disuccinimidyl carbonate (3 mmol) in DMF(˜10 mL) is added to a solution of the non-polydispersed product 48 (1mmol) in 10 mL of anydrous DMF while stirring. Sodium hydride (3 mmol)is added slowly to the reaction mixture. The reaction mixture is stirredfor several hours (e.g., 5 hours). Diethyl ether is added to precipitatethe activated oligomer. This process is repeated 3 times and the productis finally dried.

Example 40 Synthesis of Activated Hexaethylene Glycol MonomethylOligomers

[0330] The following description refers to the scheme illustrated inFIG. 10. Non-polydispersed activated hexaethylene glycol monomethylether was prepared analogously to that of non-polydispersed triethyleneglycol in Example 39 above. A 20% phosgene in toluene solution (35 mL,6.66 g, 67.4 mmol phosgene) was chilled under a N₂ atmosphere in anice/salt water bath. Non-polydispersed hexaethylene glycol 50 (1.85 mL,2.0 g, 6.74 mmol) was dissolved in 5 mL anhydrous EtOAc and added to thephosgene solution via syringe. The reaction mixture was kept stirring inthe ice bath for one hour, removed and stirred a further 2.5 hours atroom temperature. The phosgene, EtOAc, and toluene were removed byvacuum distillation, leaving non-polydispersed compound 51 as a clear,oily residue.

[0331] The non-polydispersed residue 51 was dissolved in 20 mL drydichloromethane and placed under a dry, inert atmosphere. Triethylamine(0.94 mL, 0.68 g, 6.7 mmol) and then NHS (N-hydroxy succinimide, 0.82 g,7.1 mmol) were added, and the reaction mixture was stirred at roomtemperature for 18 hours. The mixture was filtered through silica gel toremove the white precipitate and concentrated in vacuo. The residue wastaken up in dichloromethane and washed twice with cold water, twice with1 N HCl and once with brine. The organics were dried over Na₂SO₄,filtered and concentrated. Final purification was done via flashchromatography (silica gel, EtOAc) to obtain the UV activenon-polydispersed NHS ester 52.

Example 41 Synthesis of Insulin-Oligomer Conjugates

[0332] To human insulin (zinc or zinc free, 2 g, 0.344 mmol based on dryweight) in 25 mL dimethylsulfoxide (>99% purity) at 22±4° C. was added 8mL triethyl amine (>99% purity). The resulting mixture was stirred for 5to 10 minutes at 22±4° C. To the above was rapidly added the activatedoligomer of Example 18 above (0.188 g, 0.36 mmol based on 100%activation) in 7.5 mL acetonitrile under stirring at 22±4° C. Thesolution was stirred for 45 minutes and quenched with acetic acidsolution with maintaining the temperature below 27° C. The reaction wasmonitored by analytical HPLC. This reaction condition producesPEG7-hexyl-insulin, monoconjugated at the B29 position(PEG7-hexyl-insulin, B29 monoconjugated) at yield 40-60%. The crudereaction mixture (PEG7-hexyl-insulin, B29 monoconjugated, 40-60%,unreacted insulin 8-25%, related substances 15-35%) was dialyzed ordifiltered (3000-3500 molecular weight cut off, MWCO) to remove organicsolvents and small molecular weight impurities, exchanged againstammonium acetate buffer and lyophilized.

[0333] The conjugation reaction of PEG7-hexyl-insulin, monoconjugated atthe B29 position, was monitored by analytical HPLC. This analytical HPLCmethod used a Waters Delta-Pak C18 column, 150×3.9 mm I.D., 5 μm, 300 Å.The solvent system consisted of Solvent B: 0.1% TFA in 50/50methanol/water, and Solvent D: 0.1% TFA in methanol. The gradient systemwas as follows: Flow rate Time (min) % Solvent B % Solvent D (mL/min)Initial (0) 100  0 1.00 20  40 60 1.00 25 100  0 1.00

Example 42

[0334] The procedure of Example 41 is used to conjugate human insulinwith the activated oligomer of Example 24.

Example 43

[0335] The procedure of Example 41 is used to conjugate human insulinwith the activated oligomer of Example 31.

Example 44

[0336] The procedure of Example 41 is used to conjugate human insulinwith the activated oligomer of Example 37.

Example 46

[0337] The procedure of Example 41 is used to -conjugate human insulinwith the activated oligomer of Example 38.

Example 47

[0338] The procedure of Example 41 is used to conjugate human insulinwith the activated oligomer of Example 39.

Example 48

[0339] The procedure of Example 41 is used to conjugate human insulinwith the activated oligomer of Example 40.

Example 49 Determination of the Dispersity Coefficient for a Mixture ofHuman Insulin-Oligomer Conjugates

[0340] The dispersity coefficient of a mixture of human insulin-oligomerconjugates is determined as follows. A mixture of human insulin-oligomerconjugates is provided, for example as described above in Example 41. Afirst sample of the mixture is purified via HPLC to separate and isolatethe various human insulin-oligomer conjugates in the sample. Assumingthat each isolated fraction contains a purely monodispersed mixture ofconjugates, “n” is equal to the number of fractions collected. Themixture may include one or more of the following conjugates, which aredescribed by stating the conjugation position followed by the degree ofconjugation: Gly^(A1) monoconjugate; Phe^(B1) monoconjugate; Lys^(B29)monoconjugate; Gly^(A1), Phe^(B1) diconjugate; Gly^(A1), Lys^(B29)diconjugate; Phe^(B1), Lys^(B29) diconjugate; and/or Gly^(A1), Phe^(B1),Lys^(B29) triconjugate. Each isolated fraction of the mixture isanalyzed via mass spectroscopy to determine the mass of the fraction,which allows each isolated fraction to be categorized as a mono-, di-,or tri-conjugate and provides a value for the variable “M_(i)” for eachconjugate in the sample.

[0341] A second sample of the mixture is analyzed via HPLC to provide anHPLC trace. Assuming that the molar absorptivity does not change as aresult of the conjugation, the weight percent of a particular conjugatein the mixture is provided by the area under the peak of the HPLC tracecorresponding to the particular conjugate as a percentage of the totalarea under all peaks of the HPLC trace. The sample is collected andlyophilized to dryness to determine the anhydrous gram weight of thesampled. The gram weight of the sample is multiplied by the weightpercent of each component in the sample to determine the gram weight ofeach conjugate in the sample. The variable “N_(i)” is determined for aparticular conjugate (the i^(th) conjugate) by dividing the gram weightof the particular conjugate in the sample by the mass of the particularconjugate and multiplying the quotient by Avagadro's number(6.02205×10²³ mole⁻¹), M_(i), determined above, to give the number ofmolecules of the particular conjugate, N_(i), in the sample. Thedispersity coefficient is then calculated using n, M_(i) as determinedfor each conjugate, and N_(i) as determined for each conjugate.

Example 50 Purification of B29 Modified PEG7-Hexyl-Insulin,Monoconjugate, from the Crude Mixture

[0342] PEG7-hexyl-insulin, B29 monoconjugated, was purified from thecrude mixture of Example 41 using a preparative HPLC system. Lyophilizedcrude mixture (0.5 g, composition: PEG7-hexyl-insulin, B29monoconjugated, 40-60%, unreacted insulin 8-25%, related substances15-35%) was dissolved in 5-10 mL 0.01 M ammonium acetate buffer, pH 7.4and loaded to a C-18 reverse phase HPLC column (150×3.9 mm) equilibratedwith 0.5% triethylarnine/0.5% phosphoric acid buffer TEAP A). The columnwas eluted with a gradient flow using TEAP A and TEAP B (80%acetonitrile and 20% TEAP A) solvent system. The gradient system forpreparative HPLC purification of PEG7-hexyl-insulin, B29 monoconjugate,from the crude mixture was as follows: Time Flow rate (min) % TEAP A %TEAP B (mL/min) Initial (0) 70 30 30  45 64 36 30 105 60 40 30 115 40 6030 125 15 85 30 135 15 85 30

[0343] Fractions were analyzed by HPLC and the product fractions thatwere >97% purity of PEG7-hexyl-insulin, B29 monoconjugate, were pooled.The elution buffer and solvent were removed by dialysis or diafiltration(MWCO 3000-3500) against ammonium acetate buffer (0.01 M, pH 7.4) andexchanged into ammonium acetate buffer and lyophilized to produce whitepowder of PEG7-hexyl-insulin, B29 monoconjugate (purity >97%).

[0344] An analytical HPLC method using the same column and solventsystem as the method used in Example 41 to monitor the reaction was usedfor analysis of PEG7-hexyl-insulin, B29 monoconjugate. However, thegradient conditions were as follows: Time (min) % Solvent B % Solvent DFlow rate (mL/min) Initial (0) 100  0 1.00 30  10 90 1.00 35 100  0 1.00

Example 51 Cytosensor Studies

[0345] Colo 205 (colorectal adenocarcinoma cells from ATCC, catalog#CCL-222) cells that had been serum-deprived for approximately 18 hourswere suspended in 3:1 Cytosensor low-buffer RPMI-1640 media: Cytosensoragarose entrapment media and seeded into Cytosensor capsule cups at100,000 cells/10 μL droplet. Cells were allowed to equilibrate on theCytosensor to the low-buffer RPMI-1640 media at a flow rate of 100 μLper minute for approximately 3 hours until baseline acidification rateswere stable. Insulin drugs (insulin or insulin conjugates) were dilutedto 50 nM in low-buffer RPMI-1640 media and applied to the cells for 20minutes at 100 μL/minute. Following the exposure, the drug solutionswere withdrawn and the cells were again perfused under the continuousflow of low-buffer media alone. Data collection continued untilacidification rates returned to baseline levels (approximately one hourfrom application of drug solutions). The results are illustrated in FIG.14. As used in FIG. 14, insulin is human insulin; PEG4 is anon-polydispersed mixture of mPEG4-hexyl-insulin, monoconjugates; PEG10is a non-polydispersed mixture of mPEG10-hexyl-insulin, monoconjugates;PEG7 is a non-polydispersed mixture of mPEG7-hexyl-insulin,monoconjuates; PEG7_(AVG) is a polydispersed mixture ofmPEG7_(AVG)-hexyl-insulin, monoconjugates.

Example 52 Enzymatic Stability

[0346] Chymotrypsin digests were conducted in a phosphate buffer, pH7.4, at 37° C. in a shaking water bath. The insulin/insulin conjugateconcentration was 0.3 mg/mL. The chymotrypsin concentration was 2Units/mL. 100 μL samples were removed at the indicated time points andquenched with 25 μL of a 1:1 mixture of 0.1% trifluoroaceticacid:isopropyl alcohol. Samples were analyzed by reverse phase HPLC andthe relative concentrations of insulin/insulin conjugate were determinedby calculating the areas under the curves.

[0347] As used in FIG. 15, insulin is human insulin; PEG4 is anon-polydispersed mixture of mPEG4-hexyl-insulin, monoconjugates; PEG10is a non-polydispersed mixture of mPEG10-hexyl-insulin, monoconjugates;PEG7 is a non-polydispersed mixture of mPEG7-hexyl-insulin,monoconjugates; PEG7_(AVG) is a polydispersed mixture ofmPEG7_(AVG)-hexyl-insulin, monoconjugates.

Example 53 Dose Dependent Activity

[0348] An effective animal model for evaluating formulations uses normalfasted beagle dogs. These dogs are given from 0.25 mg/kg to 1.0 mg/kg ofinsulin conjugates to evaluate the efficacy of various formulations.This model was used to demonstrate that insulin conjugates according tothe present invention provide lower glucose levels in a dose dependentmanner better than polydispersed insulin conjugates, which are not partof the present invention and are provided for comparison purposes.

[0349] The protocol for dog experiments calls for a blood glucosemeasurement at time zero just before a drug is administered. Theformulation in solid oral dosage form is then inserted into the dog'smouth. Blood is drawn at 15, 30, 60 and 120 minutes and glucose levelsare measured and graphed. The lower the glucose levels, the better theactivity of the insulin conjugate. In FIG. 16, the glucose lowering, andthus the activity, of the conjugates of the present invention is shownto be dose dependent. For comparison purposes, FIG. 17 shows that theglucose lowering of polydispersed insulin conjugates in a capsuleformulation, which are not a part of the present invention, is less dosedependent than conjugates of the present invention.

Example 54 Activity and Inter-Subject Variability

[0350] An effective animal model for evaluating formulations uses normalfasted beagle dogs. These dogs are given 0.25 mg/kg of insulinconjugates to evaluate the efficacy of various formulations. This modelwas used to demonstrate that insulin conjugates according to the presentinvention provide lower inter-subject variability and better activitythan polydispersed insulin conjugates, which are not part of the presentinvention but are provided for comparison purposes.

[0351] The protocol for dog experiments calls for a blood glucosemeasurement at time zero just before a drug is administered. The oralliquid dosage formulation is then squirted into the back of the dog'smouth. In each case, the dogs received 0.25 mg/kg of this solution.Blood is drawn at 15, 30, 60 and 120 minutes and glucose levels aremeasured and graphed. The lower the glucose levels, the better theactivity of the insulin conjugate. In FIGS. 18, 19 and 20, the resultsobtained with PEG4-hexyl-insulin, monoconjugate; PEG7-hexyl-insulin,monoconjugate; and PEG10-hexyl-insulin, monoconjugate, respectively,show these PEG conjugates of the present invention result in lessinter-subject variability and higher activity than the results shown inFIG. 21 for the polydispersed PEG7_(AVG)-hexyl-insulin, monoconjugate,which is not a part of the present invention and is provided forcomparison purposes.

[0352] In the specification, there has been disclosed typical preferredembodiments of the invention and, although specific terms are employed,they are used in a generic and descriptive sense only and not forpurposes of limitation, the scope of the invention being set forth inthe following claims.

What is claimed is:
 1. A mixture of conjugates each comprising insulincoupled to an oligomer that comprises a polyethylene glycol moiety,wherein the mixture has a dispersity coefficient (DC) greater than10,000 where${D\quad C} = \frac{\left( {\sum\limits_{i = 1}^{n}{N_{i}M_{i}}} \right)^{2}}{{\sum\limits_{i = 1}^{n}{N_{i}M_{i}^{2}{\sum\limits_{i = 1}^{n}N_{i}}}} - \left( {\sum\limits_{i = 1}^{n}{N_{i}M_{i}}} \right)^{2}}$

wherein: n is the number of different molecules in the sample; N_(i) isthe number of i^(th) molecules in the sample; and M_(i) is the mass ofthe i^(th) molecule.
 2. The mixture according to claim 1, wherein thedispersity coefficient is greater than 100,000.
 3. The mixture accordingto claim 1, wherein the polyethylene glycol moiety has at least 2polyethylene glycol subunits.
 4. The mixture according to claim 1,wherein the polyethylene glycol moiety has at least 7 polyethyleneglycol subunits.
 5. The mixture according to claim 1, wherein theinsulin is human insulin and the oligomer is covalently coupled toLys^(B29) of the human insulin and has the formula:


6. The mixture according to claim 1, wherein the mixture has an in vivoactivity that is greater than the in vivo activity of a polydispersedmixture of insulin-oligomer conjugates having the same number averagemolecular weight as the mixture.
 7. The mixture according to claim 1,wherein the mixture has an in vitro activity that is greater than the invitro activity of a polydispersed mixture of insulin-oligomer conjugateshaving the same number average molecular weight as the mixture.
 8. Themixture according to claim 1, wherein the mixture has an increasedresistance to degradation by chymotrypsin when compared to theresistance to degradation by chymotrypsin of a polydispersed mixture ofinsulin-oligomer conjugates having the same number average molecularweight as the mixture.
 9. The mixture according to claim 1, wherein themixture has an inter-subject variability that is less than theinter-subject variability of a polydispersed mixture of insulin-oligomerconjugates having the same number average molecular weight as themixture.
 10. The mixture according to claim 1, wherein the oligomer iscovalently coupled to an amine function of the insulin.
 11. The mixtureaccording to claim 10, wherein the amine function is at Lys^(B29) of theinsulin.
 12. The mixture according to claim 1, wherein the conjugatecomprises a first oligomer and a second oligomer.
 13. The mixtureaccording to claim 12, wherein the first and the second oligomers arethe same.
 14. The mixture according to claim 12, wherein the firstoligomer is covalently coupled at Lys^(B29) of the insulin and thesecond oligomer is covalently coupled at N-terminal A1 or N-terminal B1of the insulin.
 15. The mixture according to claim 1, wherein theinsulin is covalently coupled to the oligomer.
 16. The mixture accordingto claim 1, wherein the insulin is covalently coupled to the oligomer bya hydrolyzable bond.
 17. The mixture according to claim 1, wherein theinsulin is covalently coupled to the polyethylene glycol moiety of theoligomer.
 18. The mixture according to claim 1, wherein the oligomerfurther comprises a lipophilic moiety.
 19. The mixture according toclaim 18, wherein the insulin is covalently coupled to the lipophilicmoiety.
 20. The mixture according to claim 18, wherein the polyethyleneglycol moiety is covalently coupled to the lipophilic moiety.
 21. Themixture according to claim 1, wherein the oligomer comprises a firstpolyethylene glycol moiety covalently coupled to the insulin by anon-hydrolyzable bond and a second polyethylene glycol moiety covalentlycoupled to the first polyethylene glycol moiety by a hydrolyzable bond.22. The mixture according to claim 21, wherein the oligomer furthercomprises a lipophilic moiety covalently coupled to the secondpolyethylene glycol moiety.
 23. The mixture according to claim 1,wherein the conjugates are each amphiphilically balanced such that eachconjugate is aqueously soluble and able to penetrate biologicalmembranes.
 24. A pharmaceutical composition comprising: the mixtureaccording to claim 1; and a pharmaceutically acceptable carrier.
 25. Amethod of treating insulin deficiency in a subject in need of suchtreatment, said method comprising: administering an effective amount ofa mixture of conjugates each comprising insulin coupled to an oligomercomprising a polyethylene glycol moiety, wherein the mixture has adispersity coefficient (DC) greater than 10,000 where${D\quad C} = \frac{\left( {\sum\limits_{i = 1}^{n}{N_{i}M_{i}}} \right)^{2}}{{\sum\limits_{i = 1}^{n}{N_{i}M_{i}^{2}{\sum\limits_{i = 1}^{n}N_{i}}}} - \left( {\sum\limits_{i = 1}^{n}{N_{i}M_{i}}} \right)^{2}}$

wherein: n is the number of different molecules in the sample; N_(i) isthe number of i^(th) molecules in the sample; and M_(i) is the mass ofthe i^(th) molecule; to the subject to treat the insulin deficiency. 26.A substantially monodispersed mixture of conjugates, each conjugatecomprising insulin coupled to an oligomer that comprises a polyethyleneglycol moiety.
 27. The mixture according to claim 26, wherein thepolyethylene glycol moiety has at least 2 polyethylene glycol subunits.28. The mixture according to claim 26, wherein the polyethylene glycolmoiety has at least 7 polyethylene glycol subunits.
 29. The mixtureaccording to claim 26, wherein at least about 96 percent of theconjugates in the mixture have the same molecular weight.
 30. Themixture according to claim 26, wherein the mixture is a monodispersedmixture.
 31. The mixture according to claim 26, wherein the mixture is asubstantially purely monodispersed mixture.
 32. The mixture according toclaim 26, wherein at least about 96 percent of the conjugates in themixture have the same molecular weight and have the same molecularstructure.
 33. The mixture according to claim 26, wherein the mixture isa purely monodispersed mixture.
 34. A substantially monodispersedmixture of conjugates each comprising human insulin covalently coupledat Lys^(B29) of the human insulin to the carboxylic acid moiety of acarboxylic acid, which is covalently coupled at the end distal to thecarboxylic acid moiety to a methyl terminated polyethylene glycol moietyhaving at least 7 polyethylene glycol subunits.
 35. The substantiallymonodispersed mixture according to claim 34, wherein the conjugates eachcomprising human insulin covalently coupled at Lys^(B29) of the humaninsulin to the carboxylic acid moiety of hexanoic acid, which iscovalently coupled at the end distal to the carboxylic acid moiety to amethyl terminated polyethylene glycol moiety having 7 polyethyleneglycol subunits.
 36. A substantially monodispersed mixture of conjugateseach comprising insulin coupled to an oligomer that comprises apolyethylene glycol moiety, said mixture having an in vivo activity thatis greater than the in vivo activity of a polydispersed mixture ofinsulin drug-oligomer conjugates having the same number averagemolecular weight as the substantially monodispersed mixture.
 37. Themixture according to claim 36, further having an in vitro activity thatis greater than the in vitro activity of the polydispersed mixture ofinsulin-oligomer conjugates.
 38. The mixture according to claim 36,further having an increased resistance to degradation by chymotrypsinwhen compared to the resistance to degradation by chymotrypsin of thepolydispersed mixture of insulin-oligomer conjugates.
 39. The mixtureaccording to claim 36, further having an inter-subject variability thatis less than the inter-subject variability of the polydispersed mixtureof insulin-oligomer conjugates.
 40. A mixture of conjugates eachcomprising insulin coupled to an oligomer that comprises a polyethyleneglycol moiety, said mixture having a molecular weight distribution witha standard deviation of less than about 22 Daltons.
 41. The mixtureaccording to claim 40, wherein the standard deviation of the molecularweight distribution is less than about 14 Daltons.
 42. The mixtureaccording to claim 40, wherein the insulin is human insulin and eacholigomer is covalently coupled to Lys^(B29) of the human insulin and hasthe formula:


43. A mixture of conjugates in which each conjugate: comprises insulincoupled to an oligomer; and has the same number of polyethylene glycolsubunits.
 44. The mixture according to claim 43, wherein the insulin ishuman insulin and each oligomer is covalently coupled to Lys^(B29) ofthe human insulin and has the formula:


45. A mixture of conjugates in which each conjugate is the same and hasthe formula: Insulin B-L_(j)-G_(k)-R-G′_(m)-R′-G″_(n)-T]_(p)   (A)wherein: B is a bonding moiety; L is a linker moiety; G. G′ and G″ areindividually selected spacer moieties; R is a lipophilic moiety and R′is a polyalkylene glycol moiety, or R′ is the lipophilic moiety and R isthe polyalkylene glycol moiety; T is a terminating moiety; j, k, m and nare individually 0 or 1; and p is an integer from 1 to the number ofnucleophilic residues on the insulin.
 46. The mixture according to claim45, wherein the polyalkylene glycol group is a polyethylene glycolmoiety.
 47. The mixture according to claim 46, wherein the polyethyleneglycol moiety has at least 2 polyethylene glycol subunits.
 48. Themixture according to claim 46, wherein the polyalkylene glycol moiety isa polyethylene glycol moiety having at least 7 polyethylene glycolsubunits.
 49. The mixture according to claim 46, wherein: R is alkyl oralkylene; R′ is polyethylene glycol having at least 7 polyethyleneglycol subunits; T is alkyl; j is 1; and k, m and n are
 0. 50. Themixture according to claim 46, wherein: B is carbonyl; R is C₅ alkylene;R′ is polyethylene glycol having 7 polyethylene glycol subunits; T ismethoxy; and k, m and n are
 0. 51. A process for synthesizing asubstantially monodispersed mixture of conjugates each conjugatecomprising insulin coupled to an oligomer that comprises a polyethyleneglycol moiety, said process comprising: reacting a substantiallymonodispersed mixture comprising compounds having the structure ofFormula I: R¹(OC₂H₄)_(m)—O⁻X⁺  (I) wherein R¹ is H or a lipophilicmoiety; m is from 1 to 25; and X⁺ is a positive ion, with asubstantially monodispersed mixture comprising compounds having thestructure of Formula II: R²(OC₂H₄)_(n)—OMs   (II) wherein R² is H or alipophilic moiety; and n is from 1 to 25, under conditions sufficient toprovide a substantially monodispersed mixture comprising polymers havingthe structure of Formula III: R²(OC₂H₄)_(m+n)—OR¹   (III); activatingthe substantially monodispersed mixture comprising polymers of FormulaIII to provide a substantially monodispersed mixture of activatedpolymers capable of reacting with an insulin drug; and reacting thesubstantially monodispersed mixture of activated polymers with insulinunder conditions sufficient to provide a substantially monodispersedmixture of conjugates each comprising insulin coupled to an oligomerthat comprises a polyethylene glycol moiety with m+n subunits.
 52. Theprocess according to claim 51, wherein R² is a fatty acid moiety or anester of a fatty acid moiety.
 53. The process according to claim 52,wherein the fatty acid moiety or the ester of a fatty acid moietycomprises an alkyl moiety at least 5 carbon atoms in length.
 54. Theprocess according to claim 51, wherein R¹ is a methyl group.
 55. Theprocess-according to claim 51, further comprising: reacting asubstantially monodispersed mixture comprising compounds having thestructure of Formula V: R²(OC₂H₄)_(n)—OH   (V) with a methanesulfonylhalide under conditions sufficient to provide a substantiallymonodispersed mixture comprising compounds having the structure ofFormula II: R²(OC₂H₄)_(n)—OMs   (II).
 56. The process according to claim55, further comprising: reacting a substantially monodispersed mixturecomprising compounds having the structure of Formula VI: R²—OMs   (VI)wherein R² is a lipophilic moiety; with a substantially monodispersedmixture comprising compounds having the structure of Formula VII:R³(OC₂H₄)_(m)—O⁻X₂ ⁺  (VII) wherein R³ is benzyl, trityl, or THP; and X₂⁺ is a positive ion; under conditions sufficient to provide asubstantially monodispersed mixture comprising compounds having thestructure of Formula VIII: R³(OC₂H₄)_(m)—OR²   (VIII); and reacting thesubstantially monodispersed mixture comprising compounds having thestructure of Formula VIII under conditions sufficient to provide asubstantially monodispersed mixture comprising compounds having thestructure of Formula V: R²(OC₂H₄)_(m)—OH   (V).
 57. The processaccording to claim 51, further comprising: reacting a substantiallymonodispersed mixture comprising compounds having the structure ofFormula IV: R¹(OC₂H₄)_(n)—OH   (IV) under conditions sufficient toprovide a substantially monodispersed mixture comprising compoundshaving the structure of Formula I: R¹(OC₂H₄)_(n)—O⁻X⁺  (I)
 58. Theprocess according to claim 51, wherein the activating of thesubstantially monodispersed mixture comprises reacting the substantiallymonodispersed mixture of polymers of Formula III with N-hydroxysuccinimide to provide an activated polymer capable of reacting withinsulin.
 59. The process according to claim 51, wherein the insulin ishuman insulin, and wherein the reacting of the substantiallymonodispersed mixture of activated polymers with a substantiallymonodispersed mixture of insulin comprises: reacting the substantiallymonodispersed mixture of activated polymers with Lys^(B29) of the humaninsulin to provide a substantially monodispersed mixture ofmonoconjugates each comprising a human insulin coupled to an oligomerthat comprises a polyethylene glycol moiety with m+n subunits.